Composition, particulate materials and methods for making particulate materials

ABSTRACT

Particulate material comprising rough mesoporous hollow nanoparticles. The rough mesoporous hollow nanoparticles may comprise a mesoporous shell, the external surface of which has projections thereon, the projections having smaller sizes than the particle size. The particulate material may be used to deliver active agents, such as insecticides and pesticides. The active agents can enter into the hollow core of the particles and be protected from degradation by sunlight. The rough surface of the particles retains the particles on plant leaves or animal hair. Methods for forming the particles are also described. Carbon particles and methods for forming carbon particles are also described.

TECHNICAL FIELD

The present invention relates to particulate materials and to methodsfor forming particulate materials. The present invention also relates toa composition. The present invention also relates to a compositioncontaining hydrophobic compounds; and/or a composition with hydrophobicproperties. Some of the particulate material may be used in compositionsin accordance with aspects of the invention.

BACKGROUND ART

Australia is among the world's largest and most successful producers ofcommercial livestock, which contributes ˜1% to Australian's grossdomestic product (GDP). The export of red meat and livestock contributeda total value of ˜$16 billion in 2012-2013. However, arthropod pestspose a serious threat to the industry. It is estimated that ticks costthe cattle industry around $170-200 million each year. Furthermore,buffalo fly and sheep lice infestations have caused millions of dollarsin losses due to the cost of implementing control strategies and lostproductivity. The high cost of ectoparasite treatment is primarily dueto the high dose rates and repeated treatments of active compoundsrequired to achieve efficacy. Moreover, many pesticides currently in usehave high toxicity, negative environmental effects and potential risksto human health and food safety. Arthropod pests are equally threateningto plant crops such as cereals, vegetables and fruit.

Spinosad is a naturally derived pesticide with low environmental impactand low mammalian toxicity. However, its use is currently limited inpart by its UV instability which reduces potency, low water solubilityand hydrophobicity, making formulation in aqueous systems difficult andhigher cost relative to conventional chemical pesticides. Spinosad iscurrently registered for use in sheep to treat lice and flyinfestations, however, its reduced potency and duration of efficacyagainst ectoparasites of cattle has prevented its registration as atreatment for buffalo fly and cattle tick. Likewise, these drawbackshave limited Spinosad's use in crop protection applications whereaqueous formulations are commonly used and UV stability is required bypesticides that reside on plant surfaces following application.

Many other compounds that are used as insecticides or pesticides arealso hydrophobic. As a result, if a water-based composition is to beused for application of those insecticidal pesticides, a suspension oremulsion will typically be required. Suspensions or emulsions can sufferfrom short shelf life, due to a tendency to separate into separatelayers. Application via spraying can also be difficult for the samereason. Further difficulties are encountered if the compounds aresensitive to light or ultraviolet light. In such circumstances, thecompounds can have a short period of effectiveness following applicationdue to the compound breaking down when exposed to sunlight.

A number of other hydrophobic compounds have beneficial effects whenused in biological systems. These compounds may include compounds havinga therapeutic effect on an animal or human (such as an antibiotic,cancer drug or other drug for treating disease), proteins and dyes foruse as marker agents. Delivery of such agents to biological systems canbe difficult.

In biological systems, hydrophobic interactions are usually consideredto be the strongest of all long-range non-covalent interactions.Hydrophobic interaction is beneficial for adsorption of biomolecules,improving interaction with cellular membranes increasing the uptake ofnanoparticles for cellular delivery as well as tailoring the releaserate of drugs. To generate nanoparticles with hydrophobic properties,the choices of hydrophobic composition or functionalization are amongthe convenient approaches. Hydrophobic material such as carbon nanotubes(CNTs) have shown great promise as nanoscaled vehicles for drugdelivery, however one of the main concerns is the fact that CNTs couldbe hazardous to environmental and human health, requiring furthersurface functionalization to reduce their intrinsic toxicity.Hydrophobic moieties such as alkanethiols and alkyl chains have beenused to modify the surfaces of various nanoparticles including gold andsilica to enhance the loading of hydrophobic drugs/protein and improvecellular delivery performance. However, chemically grafted hydrophobicgroups tended to cause unwanted toxicity and pore blocking ofnano-carriers. It is therefore a challenge to design a safe andefficient hydrophobic nanocarrier system employing an alternativeapproach.

In addition to difficulties encountered in formulating hydrophobicagents, many active molecules aside from Spinosad that are used asdrugs, insecticides or otherwise suffer from limited active lifetime inthe field due to UV degradation. This is especially the case for activemolecules that are applied topically and therefore are more likely to beexposed to UV light including topical formulations used for humans andanimals and those used in crop protection. The ability to formulatethese active molecules into a UV protecting carrier system could enablelonger duration of effectiveness.

Of course, there is little value in extending the duration of action ofan active molecule by protecting it against UV light if other factors,such as wash-off of the active molecule from the site of action, occurbefore the active molecule can take full effect. In many applicationsincluding the topical application of active molecules and in cropprotection, wash-off of active molecules by rain, wind abrasion andother erosive forces can significantly reduce the efficacy and durationof action of an active molecule.

In gene therapy, remarkable therapeutic benefits in the treatment ofdiseases caused by genetic disorders, where the efficacy of the deliveryvehicles is the key to introducing nucleic acids into cells to achievetheir functions has been demonstrated. DNA vaccination is a most recentform of treatment, where a plasmid DNA (p-DNA) encoding an antigen ofinterest is delivered into cells to induce antigen-specific immunity.Here, rather than injecting a patient with a vaccine antigen as iscommonly done in the cases of vaccination using sub-unit vaccines,patients are injected with p-DNA molecules that provide the body's cellswith the code to produce the antigen in vivo, effectively allowing thebody to produce its own antigen. Vaccination strategies using othernucleic acid forms such as messenger RNA (mRNA) are also emerging.

Effective delivery of the p-DNA into target cells has been a significantchallenge for this promising approach. DNA vaccines are promisingvaccine candidates as they are very specific, safe and well toleratedand relatively inexpensive to manufacture. However poor immunogenicityis a major problem and a significant cause of this is the inability ofthe p-DNA to be effectively delivered to the cell nucleus so that theDNA can be incorporated to then produce the vaccine antigen. Inefficientdelivery of p-DNA is caused by three main factors, all of which combinedmean that only a small proportion of p-DNA injected into the bodyactually makes it into the cell nucleus to enable the production ofvaccine antigens:

1. Breakdown of the p-DNA by nucleases after injection or delivery intothe body and before the p-DNA enters the cell2. Inability to be efficiently transported across the cell membrane intothe cell3. Inability to efficiently enter the cell nucleus once inside the cell

Delivery of p-DNA using viral delivery systems (one of the firstdelivery systems to be investigated) proved to be effective indelivering p-DNA to the cell however toxicity problems have reduce thepromise of these earlier delivery system candidates. Since then, polymermicrospheres and cationic liposomes have emerged as two promising newdelivery technologies, although neither likely is good enough to allowDNA vaccines to be widely adopted.

Cationic liposomes are able to load reasonable quantities of p-DNA andloading is easy so that the p-DNA is not damaged during the process.Protection against nucleases is good since the p-DNA can be encapsulatedwithin the liposome. However the liposomes are soft particles and so arenot very stable in vivo. Toxicity is also of great concern. Polymermicroparticles are also used as a carrier for p-DNA. The polymers aretypically more rigid than liposomes so do not have the tendency tomechanically degrade in vivo. Polymer microparticles also provide goodprotection for the p-DNA against nucleases. However the key polymersthat have been proposed (polylactic acid and poly(lactic-co-glycolicacid)) form hydrophobic particles and are negatively charged and so maynot properly encapsulate the p-DNA. In addition, the loading methods aregenerally quite harsh, which may damage the p-DNA during processing.Transfection efficiency tends to be low. Polyethylenimine (PEI) has beenshown to enable higher transfection efficiencies however these polymerscan be extremely cytotoxic. Understanding the unique loop structure ofp-DNA molecules and rational design of advanced p-DNA delivery vehiclesis highly desired for efficient gene therapy and DNA vaccinationstrategies.

It will be clearly understood that, if a prior art publication isreferred to herein, this reference does not constitute an admission thatthe publication forms part of the common general knowledge in the art inAustralia or in any other country.

SUMMARY OF INVENTION

The present invention is directed to a composition that includes one ormore hydrophobic compounds; and/or a nanostructure that has hydrophobicproperties. In other aspects of the present invention, particulatematerial and methods for forming particulate material are provided.

In a first aspect, the present invention provides particulate materialcomprising rough mesoporous hollow nanoparticles.

Rough mesoporous hollow nanoparticles are defined as hollow particles orspheres with a mesoporous shell, the external surface of which hasprojections thereon, the projections having smaller sizes than theparticle size. The particle size may range from 100 nm to 3000 nm, thesize of projections may range from 5 nm to 1000 nm, preferably from 100nm to 500 nm. In one embodiment, the projections may comprisenanospheres on the shell.

In one embodiment, the mesoporous shell may comprise, silica, Ag, Au,calcium phosphate or titanium dioxide or carbon or a carbon-basedmaterial. In one embodiment, the rough mesoporous hollow nanoparticlescomprise rough mesoporous hollow silica nanoparticles.

In one embodiment, the particles are made from a material that isnormally hydrophilic but the particles demonstrate hydrophobiccharacteristics.

The rough mesoporous hollow nanoparticles will typically have a hollowcore that is surrounded by a shell having a mesoporous structure. As theshell that surrounds and defines the hollow core is porous, compoundsmay pass through the pores and enter into the hollow core. Projectionswhich may have spherical or other shapes are present on the outside ofthe shell, providing a rough surface to the particles. Although thematerial from which the rough mesoporous hollow nanoparticles (such assilica) may normally be a hydrophilic material, the rough surfaceresults in the rough mesoporous hollow nanoparticles exhibitinghydrophobic properties, thereby allowing or even enhancing movement ofthe hydrophobic compounds into the hollow core.

The rough mesoporous hollow nanoparticles will typically have a hollowcore having a diameter of from 100 nm to 1000 nm, or from 100 nm to 700nm. The hollow core will be defined by a shell, such as a shell ofsilica in the case of rough mesoporous hollow silica nanoparticles,having a mesoporous structure. The shell (such as the shell of silica)will typically have a pore structure that includes pores in the range offrom 2 nm to 20 nm. As the shell that surrounds and defines the hollowcore is porous, compounds may pass through the pores and enter into thehollow core. The shell that surrounds the hollow core may have athickness of from 10 nm to 100 nm. The rough mesoporous hollownanoparticles may include projections or outgrowths on the surface,spaced apart from each other. The spaced projections or outgrowthsprovide surface roughness to the particles. The surface roughness issufficient to result in the rough mesoporous hollow nanoparticles takingon a hydrophobic character, in some cases extremely hydrophobic.

In embodiments where the rough mesoporous hollow nanoparticles compriserough mesoporous hollow silica nanoparticles, the rough mesoporoushollow silica nanoparticles will typically have a hollow core having adiameter of from 100 nm to 1000 nm, or from 100 nm to 700 nm. The hollowcore will be defined by a silica shell having a mesoporous structure.The silica shell will typically have a pore structure that includespores in the range of from 2 nm to 20 nm. As the silica shell thatsurrounds and defines the hollow core is porous, compounds may passthrough the pores and enter into the hollow core. The silica shell thatsurrounds the hollow core may have a thickness of from 10 nm to 100 nm.The rough mesoporous hollow silica nanoparticles may include silicaprojections or outgrowths on the surface, spaced apart from each other.The spaced silica projections or outgrowths provide surface roughness tothe particles. The surface roughness is sufficient to result in therough mesoporous hollow silica nanoparticles taking on a hydrophobiccharacter, in some cases extremely hydrophobic.

The spaced projections may comprise nanoparticles connected to the outersurface of the larger hollow nanoparticle. The nanoparticles connectedto the outer surface of the larger hollow nanoparticles may be of thesame composition as the larger hollow nanoparticles or of a differentcomposition to the larger hollow nanoparticles. The nanoparticles usedto construct the projections may have a diameter in the range of from 5nm to 100 nm and the hollow nanoparticles may have a diameter in therange of from 100 nm to 1000 nm. Alternatively, the spaced silicaprojections may comprise strands or cylinders or fibres or nodulesextending outwardly from the hollow shell of the nanoparticles. Thelength of the projections may be from 5 nm up to the diameter of thelarge hollow particle on which they reside, however they may be madelonger if required by the application. The diameter of the projectionsmay be as low as 2-3 nm or as high as 100 nm or higher and the diameteror thickness of a projection may vary along its length due to theprocess used to form it. The specific surface area of the nanoparticlesmay range from 100 m²/g to 1000 m²/g, or from 150 m²/g to 1000 m²/g, orfrom 175 m²/g to 1000 m²/g.

In embodiments where the rough mesoporous hollow nanoparticles compriserough mesoporous hollow silica nanoparticles, the rough mesoporoushollow silica nanoparticles, the spaced projections or outgrowthssuitably comprise silica projections or outgrowths. The spacedprojections of silica may comprise silica nanoparticles connected to theouter surface of a larger hollow silica nanoparticle. The silicananoparticles used to construct the projections may have a diameter inthe range of from 5 nm to 100 nm and the hollow silica nanoparticles mayhave a diameter in the range of from 100 nm to 1000 nm. Alternatively,the spaced silica projections may comprise strands or cylinders orfibres or nodules of silica extending outwardly from the hollow silicananoparticles. The length of the projections may be from 5 nm up to thediameter of the large hollow particle on which they reside, however theymay be made longer if required by the application. The diameter of theprojections may be as low as 2-3 nm or as high as 100 nm or higher andthe diameter or thickness of a projection may vary along its length dueto the process used to form it. The specific surface area of thenanoparticles may range from 100 m²/g to 1000 m²/g, or from 150 m²/g to1000 m²/g, or from 175 m²/g to 1000 m²/g.

In other embodiments, the rough mesoporous hollow nanoparticles compriserough mesoporous hollow carbon nanoparticles.

In a second aspect, the present invention provides a compositioncomprising rough mesoporous hollow nanoparticles having one or morehydrophobic materials therein or thereon.

In one embodiment of the second aspect of the present invention, thehydrophobic material comprises an insecticide or a pesticide. In apreferred embodiment, the hydrophobic material comprises Spinosad. Thehydrophobic material may be distributed throughout the particle, in theinner core, within the pores of the shell and/or on the surface of theporous shell and in between and on the projections, or any combinationof these.

As mentioned above, Spinosad is very hydrophobic, has low watersolubility and is extremely susceptible to degradation by exposure to UVlight (such as occurs when exposed to sunlight). As a result, Spinosadhas not found widespread use for treating ectoparasites and insectinfestations in livestock (such as cattle and sheep) and plants byapplying a composition containing Spinosad externally to the animal orthe plant. The present inventors have surprisingly found that roughmesoporous hollow nanoparticles, such as rough mesoporous hollow silicananoparticles, can take up Spinosad and other hydrophobic molecules in amanner that protects the hydrophobic molecules against UV lightdegradation, thereby enhancing photo stability and the duration ofinsecticidal activity. Moreover, the hollow core of the rough mesoporoushollow nanoparticles facilitates a high loading of Spinosad or otherhydrophobic molecules in the particles, allowing commercially relevantformulations to be developed. In addition, the hollow and rough surfacemorphology of the rough mesoporous hollow nanoparticles increases thehydrophobicity of the particles and further enhances Spinosad loadingcapacity. The rough mesoporous hollow nanoparticles have also been foundto adhere more strongly to skin, hair and other surfaces such as theleaves of plants, thus further prolonging the duration of insecticidalactivity of Spinosad under field conditions. The rough mesoporous hollownanoparticles are likely to adhere more strongly to leaves of plants,particularly leaves that have hairs on them. This improved adhesionfurther enhances the potency and longevity of the insecticide byminimising wash-off of the insecticide residues following application.Consequently, more environmentally friendly formulations where a lowerlabel dose is used may be feasible with the present invention.

The present invention is not limited to compatibility with anyparticular class of hydrophobic molecule, and as such, may be used witha wide range of hydrophobic molecules. Other hydrophobic pesticides thatmay be formulated with the particles of the present invention include,but are not limited to pyrethroid, azadirachtin (neem oil) andpyrethrum. Similar to Spinosad, these are natural products which aresafe to use but breakdown quickly under sunlight. Indeed, many newpharmaceutically active molecules currently under development sufferfrom problems of hydrophobicity or UV degradation and these are likelyto be compatible with the particles of the present invention.

In one embodiment, the present invention provides an insecticidalcomposition for external application to an animal or plant, thecomposition comprising rough mesoporous hollow nanoparticles having oneor more hydrophobic insecticidal materials therein. In one embodiment,the rough mesoporous hollow nanoparticles comprise rough mesoporoushollow silica nanoparticles.

The one or more hydrophobic materials will suitably be present in thehollow core of the nanoparticles. The one or more hydrophobic materialsare also likely to be present in the spaces between raised or projectingregions that produce the surface roughness of the nanoparticles.

The rough mesoporous hollow nanoparticles can also be used as efficientvehicles for delivery of hydrophobic material to biological systems,such as drug delivery of hydrophobic drugs, carriers and delivery agentsfor hydrophobic proteins and as carriers and delivery agents forhydrophobic dyes that can be used as marker agents. The rough mesoporoushollow nanoparticles after further modification with hydrophobiccompounds to yield superhydrophobic particles can be employed in theremoval of water pollutants and in surface coatings for self-cleaningapplications. Methods for hydrophobic modification of surfaces such assilica including the covalent attachment of moieties containinghydrophobic groups using silanes and other agents are well-known tothose skilled in the art.

In one embodiment, the hydrophobic material may comprise a hydrophobicprotein, such as RNase A, insulin or lysozyme, a hydrophobic dye, suchas disperse red 1, solvent red or rose bengal, a hydrophobic drug ortherapeutic agent, such as griseofluvin, curcumin, ibuprofen orerythromycin or vancomycin or an essential oil such as oregano oil. Inthe case of essential oils, the present invention can provide a meansfor increasing the solubility of the hydrophobic essential oils, makingthem more bioavailable and therefore enabling dose sparing strategies tominimise essential oil costs in the manufacture of formulations.Essential oils are also known to be relatively volatile compounds andsignificant amounts of oil can be lost to evaporation during themanufacture, storage and use of essential oil formulations. By loadingthe essential oils into the particles of the present invention, lossesto evaporation can be minimised and essential oils costs inmanufacturing a formulation can be reduced without negatively affectingthe efficacy of the product. Lysozyme and other enzymes that are used incosmetics, animal feed supplements and other applications may also beformulated with the particles of the present invention. Here, theparticles can provide a slow release function, which in the case oflysozyme for example which has antibacterial properties, can result insustained suppression of bacteria over time. During the manufacture andstorage of enzyme formulations, many enzymes suffer degradation as aresult of thermal breakdown, hydrolysis or otherwise, requiring excessenzyme to be used in formulations in order to compensate for these yieldlosses. For example, in the steam pelleting process used to make someanimal feeds, the application of steam can result in denaturation ofsome of the enzyme content, requiring either excess enzyme to be addedto the formulation or the use of expensive equipment to spray enzymeonto the resulting pellets following the steam pelleting process.Formulation of enzymes with the particles of the present invention canprotect the enzyme from degradation. These active ingredients may beloaded into the internal cavity provided by the particles, on theoutside of the particles entangled with the projections or a combinationof both. How the active ingredient is distributed between the internalcavity and external surface depends of the desired rate of release, thesize of the molecule, the desired loading level, the extent ofprotection needed by the active ingredient and other factors.

Without wishing to be bound by theory, the present inventors havepostulated that active molecules may adsorb onto the surface of theparticles or be inserted into the voids between the projections on theparticles and this provides a degree of protection against degradationof the active materials, even if the active materials do not enter(partially or completely) into the hollow core of the particles.

The present inventors have also found that rough mesoporous hollownanoparticles can be used to provide for sustained release of compoundstaken up therein. Accordingly, in a third aspect, the present inventionprovides a composition for providing sustained release of a compound,the composition comprising rough mesoporous hollow nanoparticles havingcompounds taken up therein. In this aspect, the compound may be ahydrophobic compound or may be a hydrophilic compound. The compound maycomprise any of the materials described as being suitable for use in thefirst aspect of the present invention. The compound may be a therapeuticagent, such as an antibiotic. The antibiotic may be, for example,vancomycin or metronidazole.

In other aspect, compatibility of the present invention is not limitedto use with hydrophobic molecules. Many hydrophilic molecules couldbenefit from advantages provided by the particles of the presentinvention such as slow release, protection against UV degradation andenhanced adhesion to plant, animal or other surfaces.

Accordingly, in a fourth aspect, the present invention provides acomposition comprising rough mesoporous hollow nanoparticles having oneor more hydrophilic materials therein.

In a fifth aspect, the present invention may also relate to acomposition comprising rough mesoporous hollow nanoparticles having oneor more active molecules therein or thereon. In some embodiments, theactive molecules may be any of the active molecules described herein.

The present inventors have also found that the particles of the presentinvention can function as an effective delivery system for nucleic acidssuch as plasmid DNA (p-DNA) and messenger RNA (mRNA) that are used inemerging vaccination strategies. In the case of p-DNA, it is desirableto be able to protect the p-DNA molecule from attack by nucleases onentry of the p-DNA into the body. This mode of degradation of p-DNA isresponsible for a significant reduction in the efficacy of DNA vaccines.Due to the large size of the p-DNA molecules, when formulated with theparticles of the present invention p-DNA is largely distributed on theoutside of the particles, secured by the projections on the surface ofthe particles. This is sufficient to provide a high degree of protectionagainst attack by nucleases. In formulating a DNA or mRNA-based vaccine,the particles of the present invention may be coated with substancesthat increase the affinity of the particles to these nucleic acids. Thismay involve covalently grafting chemical functional groups onto theparticles, or applying a coating that interacts with the particlesurface via hydrogen bonding, electrostatic attraction or some othermeans known to those skilled in the art. For example, polyethylenimine(PEI) may be coated onto the particles. With a formulation substantiallystable against attack by nucleases, the next challenge for a DNA vaccinedelivery system is to efficiently cross the cell membrane carrying thep-DNA. The size of the particles of the present invention is well-suitedfor efficient cellular uptake by host cells after forming complexes withp-DNA, mRNA, siRNA or other nucleic acids. In some instances, where thenucleic acid molecules are located on the outside of the shell, securedto the particles via entanglement in the projections, it may not benecessary to use a shell with any porosity since the active molecules donot substantially enter the internal cavity.

Accordingly, in a sixth aspect, the present invention provides acomposition comprising rough nanoparticles at least partially coatedwith one or more nucleic acids. The rough nanoparticles may have littleor no porosity. The rough nanoparticles may have a solid core or theymay have a hollow core. The rough nanoparticles may have a mesoporousstructure but, due to the size of the one or more nucleic acids, theremay be little or no penetration of the pores of the nanoparticle by theone or more nucleic acids.

In a seventh aspect, the present invention provides particulate materialcomprising rough nanoparticles comprising a core, the external surfaceof which has projections thereon, the projections having smaller sizesthan the particle size, the rough nanoparticles having a particle sizeranging from 100 nm to 3000 nm, a size of the projections ranging from 5nm to 1000 nm.

In this aspect, the size of the projections may range from 100 nm to 500nm. The projections may comprise nanospheres on the shell or outgrowthson the shell. The core may comprise silica, Ag, Au, calcium phosphate ortitanium dioxide or carbon or a carbon-based material. The nanoparticlesmay have a core having a diameter of from 100 nm to 1000 nm. The coremay be a solid core or a hollow core. The nanoparticles have little orno porosity.

In an eight aspect, the present invention provides a compositioncomprising rough nanoparticles as claimed in any one of claims 79 to 85at least partially coated with nucleic acids. The nucleic acid may beselected from one or more of plasmid DNA (p-DNA) and messenger RNA(mRNA).

In a ninth aspect, the present invention provides use of roughmesoporous hollow nanoparticles in accordance with the seventh aspect ofthe present invention for vehicles for delivery of hydrophobic materialto biological systems.

In a tenth aspect, the present invention provides use of roughmesoporous hollow nanoparticles in accordance with the seventh aspect ofthe present invention for drug delivery of hydrophobic drugs, or ascarriers and delivery agents for hydrophobic proteins or as carriers anddelivery agents for hydrophobic dyes that can be used as marker agents.

In an eleventh aspect, the present invention provides use of roughmesoporous hollow nanoparticles in accordance with the seventh aspect ofthe present invention for removal of water pollutants or in surfacecoatings for self-cleaning applications. The particles may be modifiedwith hydrophobic compounds to yield superhydrophobic particles.

In a twelfth aspect, the present invention provides a method for formingrough nanoparticles comprising the steps of forming a particle from areaction mixture, the particle being formed from a first material,adding a precursor of a second material to the reaction mixture to forma shell of the second material around the particle, the shell havingoutgrowths of the second material extending therefrom with firstmaterial being formed from the reaction mixture between the outgrowthsof the second material and subsequently removing the first materiallocated exteriorly to the shell. The shell may comprise a solid shellhaving little or no porosity. The step of removing the first materiallocated exteriorly to the shell may leave a core of first materialinside the shell.

In some embodiments, the nucleic acid may be plasmid DNA or mRNA. Two ormore nucleic acids may be used.

Where the nucleic acid comprises plasmid DNA, the composition maycomprise a DNA vaccine composition.

In one embodiment of the present invention, rough mesoporous hollownanoparticles may be prepared by forming a hollow shell nanoparticle andadding nano particles with smaller sizes onto the hollow shellnanoparticles of relatively larger size so that the smaller particlesform outgrowths or projections on the outer surface of the larger hollowshell. The hollow silica nanoparticles may be mesoporous. According tothis approach, the particles that will form the projections may besynthesised separately to the larger hollow shells.

In one embodiment of the present invention, rough mesoporous hollowsilica nanoparticles may be prepared by forming a hollow silica shellnanoparticle and adding silica nano particles with smaller sizes ontothe hollow silica shell nanoparticles of relatively larger size so thatthe smaller silica particles form outgrowths or projections on the outersurface of the larger hollow silica shell. The hollow silica shellnanoparticles may be mesoporous. According to this approach, the silicaparticles that will form the projections may be synthesised separatelyto the larger hollow silica shells.

In another embodiment, the rough mesoporous hollow nanoparticles may beformed by forming a sacrificial particle from a reaction mixture, thesacrificial particle being formed from a carbon-based material, adding ashell material precursor to the reaction mixture to form a porous shellaround the sacrificial particle, the shell having outgrowths of materialcontaining silicon extending therefrom with carbon-based material beingformed from the reaction mixture and being deposited between theoutgrowths of material and subsequently removing the carbon-basedmaterial. Here, the outgrowth material or outgrowth material precursorand carbon-based material are co-deposited onto the porous shell in aspatially inhomogeneous manner such that subsequent removal of thecarbon-based material leaves projections of material protruding from thesurface of the shell. The carbon-based material co-deposited with theprojections may be deposited from the carbon-based precursor left overfrom the formation of the sacrificial particles or carbon-basedprecursor may be subsequently added to the mixture. It is believed thatthis fabrication method is unique.

In another embodiment, the rough mesoporous hollow silica nanoparticlesmay be formed by forming a sacrificial particle from a reaction mixture,the sacrificial particle being formed from a carbon-based material,adding a silica precursor to the reaction mixture to form a porous shellcontaining silicon around the sacrificial particle, the shell containingsilicon having outgrowths of material containing silicon extendingtherefrom with carbon-based material being formed from the reactionmixture between the outgrowths of material containing silicon andsubsequently removing the carbon-based material. Here, silicon andcarbon-based material are co-deposited onto the porous silicon shell ina spatially inhomogeneous manner such that subsequent removal of thecarbon-based material leaves projections of silicon protruding from thesurface of the silica shell. The carbon-based material co-deposited withthe silicon projections may be deposited from the carbon-based precursorleft over from the formation of the sacrificial particles orcarbon-based precursor may be subsequently added to the mixture.

Accordingly, in a thirteenth aspect, the present invention provides amethod for forming rough mesoporous hollow nanoparticles comprising thesteps of forming a sacrificial particle from a reaction mixture, thesacrificial particle being formed from a first material, adding aprecursor of a shell material to the reaction mixture to form a shell ofa second material around the sacrificial particle, the shell havingoutgrowths of material extending therefrom with first material beingformed from the reaction mixture between the outgrowths of the secondmaterial and subsequently removing the first material.

In one embodiment of this method, the first material is a carbon-basedmaterial and the second material is a silicon or silica-based material.In this embodiment, the method for forming rough mesoporous hollownanoparticles comprising the steps of forming a sacrificial particlefrom a reaction mixture, the sacrificial particle being formed from acarbon-based material, adding a precursor of a shell material to thereaction mixture to form a shell around the sacrificial particle, theshell having outgrowths of material extending therefrom withcarbon-based material being formed from the reaction mixture between theoutgrowths of material and subsequently removing the carbon-basedmaterial. The sacrificial particle can be made from variouspolymerisation precursors, e.g. aminophenol-formaldehyde or dopamine.

In one embodiment, the carbon-based material comprises a polymer formedby the reaction of two or more monomers or polymer precursors. In oneembodiment, the shell containing silicon and the outgrowths of materialcontaining silicon comprise silica. In this embodiment, the silicaprecursor forms a silica shell around the sacrificial particle withoutgrowths of silica extending therefrom.

In one embodiment, the silica precursor material forms silica at afaster rate than the formation of the carbon containing material. As aresult, a shell of silica is first deposited on the surface of thepreformed sacrificial particles. Typically, once the shell of silica hasbeen formed, sufficient time has passed for the precursors to thecarbon-based material to start forming additional carbon-based material.Therefore, the growth of carbon-based material competes with the growthof silica species on the shell of silica, which results inpreferentially vertical outgrowths of the respective species. Thisresults in the formation of a layer of “rod-like” silica projections andcarbon-based material between the projections. When the silica speciesin the reaction mixture are consumed, the remaining precursors to thecarbon-based material further deposit or react to form an outermostlayer of carbon-based material. The carbon-based material may be removedby any suitable process, typically by heating, such as calcination, orby using an appropriate solvent. This method may also be used withparticles made from materials other than silica, such as Ag, Au, calciumphosphate and titanium dioxide.

In instances where it is desired to form a nanoparticle having a solidcore, the step of removing the first material may be controlled so thatthe core material is not removed. In instances where it is desired toform a particle having little or no porosity, the shell around the coreis formed so that it is a shell having little or no porosity.

In one embodiment, the carbon-based material is formed from a reactionmixture that comprises resorcinol-formaldehyde, aminophenol-formaldehydeor dopamine. The sacrificial particles may be formed under typicalStöber synthesis conditions of ammonia aqueous solution, deionized waterand ethanol with of pH=11.5 at room temperature. The weight ratio ofsilica precursor of TEOS to resorcinol and formaldehyde is typically1:0.71 and 1:0.81, respectively. The silica precursor may comprisetetraethyl orthosilicate (TEOS), tetrapropyl orthosilicate (TPOS) ortetrabutoxysilane (TBOS), tetramethyl orthosilicate (TMOS) or othersilica precursors known to those skilled in the art. Under the reactionconditions used, the silica precursor may form silica. Alternatively,the silica precursor may form a silicon containing material that may besubsequently converted to silica.

In one embodiment of the thirteenth aspect of the present invention, thesilica precursor is added to the reaction mixture and a further additionof precursors for the carbon-based material is subsequently made at alater time.

In one embodiment, one or more of the precursors for the carbon-basedmaterial in the reaction mixture are essentially fully consumed informing the sacrificial particle, following which the precursor for theshell material is added and further of the precursors for thecarbon-based material are added a predetermined period after addition ofthe shell material precursor. This allows the shell to form around thesacrificial particle. This shell will surround the hollow core in thefinal rough mesoporous hollow nanoparticle. In another embodiment, theshell material precursor forms material at a significantly faster ratethan the precursors for the carbon-based material. This will also resultin the formation of a shell around the sacrificial particle. However,formation of the carbon-based material from its precursors will stilloccur and this will tend to occur on the surface of the shell or siliconcontaining shell in competition with the deposition of further shellmaterial. As a result, separate islands of carbon-based material andshell material will form on the surface of the shell. Further depositionof the carbon-based material will tend to occur on the islands ofcarbon-based material, leading to outgrowths of carbon-based material.Similarly, further deposition of the shell material will tend to occuron the islands of shell material, leading to outgrowths of the shellmaterial. Thus, rod-like outgrowths of each material will occur. Oncethe shell material precursor has been exhausted, further carbon-basedmaterial will be deposited to form an outer shell of carbon-basedmaterial. Removal of the carbon-based material, such as by calcinationin air, results in the formation of the rough mesoporous hollownanoparticles.

In one embodiment in which the rough mesoporous hollow nanoparticlescomprise rough mesoporous hollow silica nanoparticles one or more of theprecursors for the carbon-based material in the reaction mixture areessentially fully consumed in forming the sacrificial particle,following which the silica precursor is added and further of theprecursors for the carbon-based material are added a predeterminedperiod after addition of the silica precursor. This allows the silica orsilicon containing shell to form around the sacrificial particle. Thissilica or silicon containing shell will surround the hollow core in thefinal rough mesoporous hollow silica nanoparticle. In anotherembodiment, the silica precursor forms silica or silicon containingmaterial at a significantly faster rate than the precursors for thecarbon-based material. This will also result in the formation of asilica shell or silicon containing shell around the sacrificialparticle. However, formation of the carbon-based material from itsprecursors will still occur and this will tend to occur on the surfaceof the silica shell or silicon containing shell in competition with thedeposition of further silica or silicon containing material. As aresult, separate islands of carbon-based material and silica/siliconcontaining material will form on the surface of the silica/siliconcontaining material shell. Further deposition of the carbon-basedmaterial will tend to occur on the islands of carbon-based material,leading to outgrowths of carbon-based material. Similarly, furtherdeposition of the silica/silicon containing material will tend to occuron the islands of silica/silicon containing material, leading tooutgrowths of silica/silicon containing material. Thus, rod-likeoutgrowths of each material will occur. Once the silica precursor hasbeen exhausted, further carbon-based material will be deposited to forman outer shell of carbon-based material. Removal of the carbon-basedmaterial, such as by calcination in air, results in the formation of therough mesoporous hollow silica nanoparticles.

The amount of shell material precursor that is added to the reactionmixture may be controlled to control the thickness of the shell, theporosity of the shell and the spacing between the outgrowths. In someembodiments, the shell that is formed on the surface of the sacrificialparticle may comprise a discontinuous shell having gaps or spacestherein. Indeed, in some embodiments, the shell may comprise adiscontinuous material layer or a relatively continuous interlinkedmaterial layer.

The reaction conditions and reaction time may be controlled in order tocontrol the size of the sacrificial particle that is first formed. Thiswill, of course, allow for control of the size of the hollow core of thefinal rough mesoporous hollow nanoparticles. It will be appreciated thatthe shell that defines the hollow core of the final rough mesoporoushollow nanoparticles may shrink during the step of removing thecarbon-based material.

In a fourteenth aspect, the present invention provides a method forforming carbon nanoparticles comprising the steps of forming a reactionmixture containing a silica precursor and one or more precursors ofcarbon-based material wherein silica or silicon containing particles areformed and carbon-based materials form on the silica or siliconcontaining particles to thereby form a shell of carbon-based material onthe silica or silicon containing particles, adding further silicaprecursor to the reaction mixture to form further silica or siliconcontaining material on the shell of carbon-based material, whereinfurther carbon-based material is formed and deposits between and overthe further silica or silicon containing material, and removing thesilica or silicon containing material to thereby obtain carbonnanoparticles. The silica or silicon containing material could bereplaced by other materials, e.g. titanium dioxide derived fromaluminium isopropoxide or aluminium oxide from titanium (IV) butoxide.The obtained nanoparticles can be N-doped compositions of carbonnanoparticles by replacing RF with aminophenol-formaldehyde or dopaminecontaining N as polymerisation precursors in alcohol-water system.

In a fifteenth aspect, the present invention provides a method forforming carbon nanoparticles comprising the steps of forming a reactionmixture containing a precursor of a first material and one or moreprecursors of carbon-based material wherein particles of the firstmaterial are formed and carbon-based materials form on the particles offirst material to thereby form a shell of carbon-based material on theparticles of first material, adding further first material precursor tothe reaction mixture to form further first material on the shell ofcarbon-based material, wherein further carbon-based material is formedand deposits between and over the further first material, and removingthe first material to thereby obtain carbon nanoparticles.

In one embodiment, the carbon-based material is carbonised. Thecarbon-based material may be carbonised before removal of the firstmaterial. In one embodiment, the particle is subjected to a hydrothermaltreatment prior to the carbonisation step.

The carbon nano particles formed in the method of the eighth and ninthaspects of the invention comprise mesostructured hollow carbon sphereshaving a bilayered structure. By controlling the thickness of thecarbon/silica or carbon/first material shells, the bilayered morphologyof the particles and the mesopore size can be regulated. The bilayeredmorphology may comprise invaginated, endo-invaginated or intact spheres.The diameter of the carbon nanoparticle and hollow core size may becontrolled to range from 100-1000 nm, the thickness of the wallsurrounding the hollow core can be adjusted from 5-100 nm. The porevolume and surface area of the bilayered carbon nanoparticles may be inthe range of 1-3 cm³ g⁻¹ and 800-1300 m² g⁻¹, respectively.

In a sixteenth aspect, the present invention provides carbon particlescomprising comprise mesostructured hollow carbon spheres having abilayered structure. The bilayered morphology may comprise invaginated,endo-invaginated or intact spheres. The diameter of the carbonnanoparticle and hollow core size may range from 100-1000 nm, thethickness of the wall surrounding the hollow core may range from 5-100nm. The pore volume and surface area of the bilayered carbonnanoparticles may be in the range of 1-3 cm³ g⁻¹ and 800-1300 m² g⁻¹,respectively. The bilayered structure may comprise two spaced partial orcomplete carbon shells, with the inner shell being essentially hollow.The carbon particles may have a multi-layered structure, having 2 ormore spaced partial or complete carbon shells.

In the field of energy storage, the desire to achieve higher energydensities is driving investigation of new high capacity electrodematerials. However, unlike established materials such as graphite asused in lithium ion batteries, some of these promising high capacitycandidate materials suffer from poor electronic conductivity and in somecases their cycling involves significant volume changes. Theselimitations can result in poor power capability and cycle liferespectively. The inventors of the present invention have found that thecarbon nanoparticles of the present invention can be used as a carrieror encapsulant for electrode materials that suffer from thesechallenges. Battery active materials may be loaded into the carbonparticles which are inherently good conductors of electrons such thatthe active material is located within the internal cavity, in betweenthe carbon walls, on the outside of the particles or any combination ofthese locations. By being in very close contact with the carbonparticle, electronic conductivity challenges of the active material areminimised. In addition, the encapsulation of the active materialconfines it and restricts movement and subsequent loss of activematerial from the electrode as a result of volume changes duringcycling, resulting in improved cycle life for the battery. Thecomposition of battery active materials that may be used in the presentinvention include those materials that suffer from poor electronicconductivity and poor cycle life. These materials are well known tothose skilled in the art and include sulfur and sulfur derivatives suchas selenium sulfide (SeS₂). Other electrode active materials may includesulphur and sulphur containing compounds, silicon and mixturescontaining silicon, tin and tin-containing alloys and mixtures, antimonyand antimony-containing alloys and mixtures or any combination of these.Indeed, the present invention emcompasses any material known to besuitable for use as such by the person skilled in the art.

Accordingly, in a further aspect, the present invention provides amaterial for use in a battery or other electric power storage devicecomprising carbon particles as described above loaded with one or moreelectrode active materials.

By “electrode active electric materials” we mean a material that canaccept electric charge to reach a charged state and subsequentlydischarge electricity to move toward a discharged state.

In embodiments of this aspect of the present invention, the material maybe used as a battery electrode material, in a battery electrode, or in abattery cell, or in a capacitor, supercapacitor or a pseudo capacitor,or in an electrochromic device, or indeed in any application where useof a material that can be charged and discharged is required.

In one embodiment of the seventh, eighth or ninth aspects of theinvention, the silica precursor comprises tetraethyl orthosilicate(TEOS). The precursors for the carbon-based material may compriseresorcinol and formaldehyde.

In one embodiment, the silica or silicon containing material is removedby etching or by dissolution. For example, the silica or siliconcontaining material may be removed by etching or dissolving in HF (5%)aqueous solution or sodium hydroxide (1M) solution.

In one embodiment, the invention provides a composition comprising aninsecticidal composition for external application to an animal or plant,the composition comprising rough mesoporous hollow nanoparticles havingone or more hydrophobic insecticidal materials therein or thereon, therough mesoporous hollow nanoparticles comprise rough mesoporous hollowsilica nanoparticles, the one or more hydrophobic materials beingpresent in the hollow core of the nanoparticles and/or in the spacesbetween raised or projecting regions that produce the surface roughnessof the nanoparticles.

Any of the features described herein can be combined in any combinationwith any one or more of the other features described herein within thescope of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the invention will be described with reference tothe following drawings, in which:

FIG. 1 shows a schematic diagrams of (a) MSHSs-RS and (b) MSHSs-SS,where (b) shows the porous shell surrounding the inner cavity, R2represents the radius of the cavity, R1 represents the radius ofspherical projections on the surface of the shell and white areasbetween the spherical projections show the presence of air whenparticles are immersed in water, providing some hydrophobic character tothe particles. SEM (c and d), TEM images (e and f) of MSHSs-RS andMSHSs-SS;

FIG. 2 shows optical images (phase contrast) of (A) kangaroo fur, (B)silica nanoparticles MSHSs-RS, kangaroo fur treated with (C, E) MSHSs-SSand (D, F) MSHSs-RS (E and F are samples washed by water);

FIG. 3 shows FTIR spectra of a series of samples including purespinosad, nano-spinosad-X, silica nanoparticles and the physical mixtureof spinosad and silica nanoparticles.

FIG. 4 shows (A) TGA profiles and (B) DSC profiles of (black) purespinosad, (red) nano-spinosad-0.4, (blue) nano-spinosad-0.5, (d)nano-spinosad-0.6. (pink in B is the DSC curve for physical mixture ofspinosad and silica nanoparticles);

FIG. 5 shows wide angle XRD patterns of a series of samples includingpure spinosad, nano-spinosad-X, silica nanoparticles and the physicalmixture of spinosad and silica nanoparticles;

FIG. 6 shows FE-SEM images of pure silica nanoparticles in (A) low and(B) high magnifications, (C) pure spinosad, (D) nano-spinosad-0.4, (E)nano-spinosad-0.5 and (F) nano-spinosad-0.6;

FIG. 7 shows time dependent release profiles of pure spinosad andnano-spinosad;

FIG. 8 shows HPLC patterns of pure spinosad and nano-spinosad after UVirradiation;

FIG. 9 shows a schematic illustration of the synthesis procedures ofmonodispersed rough silica hollow spheres in accordance with anembodiment of the third aspect of the present invention, as described inexample 2;

FIG. 10 shows TEM images (A, B, C) and DLS measurement (D) of roughsurface silica hollow spheres S-1.4, S-1.0 and S-1.2 made in example 2;

FIG. 11 shows Electron Tomography slices of the rough surface silicahollow spheres S-1.4 (A), S-1.0 (B), S-0.6 (C);

FIG. 12 shows N₂ sorption isotherm (A) and pore size distribution by BJHadsorption branch (B) of the rough silica hollow spheres made in example2;

FIG. 13 shows SEM images for the contact angle test of smooth silicahollow sphere (A), S-1.4 (B), S-1.0 (C) and S-0.6(D). Insert are the TEMimages for its corresponding particles;

FIG. 14 shows lysozyme adsorption capacity of smooth and rough silicahollow spheres;

FIG. 15 shows the uptake and release behaviour of the nanoparticlestowards hydrophobic and hydrophilic molecules. a) Loading capacity ofMHS and RMHS on drug and different proteins, b) uptake rate of DR1; Thesolutions containing particles were pre-treated with sonication beforeadding the proteins or drugs for loading; c) the release behaviour ofVAN for 400 nm particles and d) The release behaviour of VAN for 200 nmparticles. The error bars reflect the standard deviation of themeasurements.

FIG. 16 shows antibacterial performance. a) Dose dependent antibacterialactivity against E. coli of RMHS200-VAN, MHS200-VAN and free VANcultured for 18 h, using PBS as a control. b) Time dependentantibacterial study at the VAN dosage of 25 mg ml⁻¹ up to 24 h. c) TEMimages of E. coli treated in PBS, d) E. coli treated in VAN, e) E. colitreated in MHS200-VAN and f) E. coli treated in RMHS200-VAN at thedosage of 25 mg m⁻¹ for 18 h. * indicated 100% inhibition. The errorbars reflect the standard deviation of the measurements. Scale bar=500nm (see example 3).

FIG. 17 shows a schematic illustration for the synthesis of invaginated,endo-invaginated and intact MHCSs through a sequential heterogenousnucleation mechanism in accordance with an embodiment of the fourthaspect of the invention;

FIG. 18 shows SEM (A, C) and TEM (B, D) images of invaginated and intactMHCSs, respectively. Digital images (E) of two MHCSs dispersed inaqueous solutions showing the Tyndall effect and the particle sizedistribution curves (F) by DLS measurement;

FIG. 19 shows ET slides of invaginated MHCS (A) and intact MHCS (C), ETreconstruction of invaginated MHCS (B) and intact MHCS (D). Scale barsare 100 nm;

FIG. 20 shows particle sizes of pure silica (curve I), pure RF (curveII), silica@RF (curve III) and silica@RF@silica@RF (curve IV) as afunction of reaction time;

FIG. 21 shows MHCS with inner shell invaginated and outer shell intact.Diagram (A) shows the internal structure of the particle relative to theXYZ orientation. Tilted TEM images (B and C). Slice (D) cuts the YZplane in the centre of the particle while silde(E) and slice (F) cut theXY plane at position a and b as indicated in diagram (A), respectively.Scale bars are 100 nm;

FIG. 22 shows SEM images of S-SHSs (a), R-MSHSs-B (b) and R-MSHSs (c)adhered on E. coli surface, and quantitative analysis of silica contentadhered on bacteria from ICP-OES (d), as described in Example 4;

FIG. 23 shows (a) Lysozyme loading, (b) time dependent antibacterialactivities of free lysozyme and lysozyme loaded silica particles at thelysozyme dosage of 700 ng/mL, as described in example 5;

FIG. 24 shows (a) TEM images of MSHS-RS particles made using resorcinol0.15 g with particle diameter of 307±18 nm, (b) resorcinol 0.30 gparticles with particle diameter of 564±20 nm, (c) resorcinol 0.45 gparticles with particle diameter of 704±25 nm and (d) resorcinol 0.60 gparticles with particle diameter of 837±35 nm (diameter measured from 20particles), as described in example 8; and

FIG. 25 shows TEM image of (a) as-prepared carbon particles and (b)SeS₂/carbon composite (inset: line scanning EDX), and (c) cyclingperformance of pure SeS2 and SeS₂/carbon composite at 200 mA/g.

DESCRIPTION OF EMBODIMENTS

Throughout the examples, the following abbreviations will be used:

MSHS—mesoporous silica hollow spheres (having a relatively smoothsurface).

MSHS-RS—mesoporous silica hollow spheres with a rough surface.

Example 1

Development of a Nano-Pesticide with Improved Safety and Performance.

Ticks and buffalo fly cause over $400 million/year in economic losses tothe Australian livestock industry and are currently treated with highlytoxic synthetic pesticides. Spinosad, a naturally derived pesticide withlow environmental impact and low toxicity will be loaded into silicahollow spheres which will improve adhesion to skin/hair and protectagainst UV degradation. The nano-spinosad pesticide will have enhancedefficacy and effective duration in field conditions compared toconventional pesticides, significantly reducing the cost of pestcontrol.

Spinosad was supplied by the Elanco Animal Health. Mesoporous silicahollow spheres with a rough surface (MSHSs-RS) and a smooth surface(MSHSs-SS) were synthesized in Yu Group in Australian Institute forBioengineering and Nanotechnology, University of Queensland. Kangarooskin samples with fur were purchased from Skinny Shop, Australia as ananimal model. The skin samples were washed thoroughly by distilled waterand cut into small pieces at 1 cm² before the tests. All the otherreagents were of analytical reagent grade.

Adhesive Property of Nanoparticles on Animal Skin Fur

The adhesive behaviour of the nanoparticles on animal fur was evaluatedon a treated kangaroo skin with fur as an animal model. Silicananoparticles (2 mg/cm²) were dispersed in an ethanol solution and thesolution was dripped homogenously on the fur side of the kangaroo skinpieces (1 cm²). Skin pieces were then allowed to dry at 40° C.overnight. The attachment of the nanoparticles on hair was observed byconfocal microscopy (LSM ZEISS 710) before and after several washingswith water. Pure skin samples and silica nanoparticles (using MSHSs-RSas an example) were also observed under the microscope. Quantitativeamount of the attached nanoparticles before and after washing weremeasured and compared by inductively coupled plasma optical emissionspectrometry (ICPOES, a Vista-PRO instrument, Varian Inc, Australia).Skin samples containing the nanoparticles were dissolved in 2M NaOHovernight under stirring to allow dissolution of the silicananoparticles and silicon concentration were measured. Silicon amount ofsimilar size skin without nanoparticles were also measured as the blank.

Preparation of Nano-Spinosad

A rotary evaporation method was utilized for encapsulation of spinosadinto the silica nanoparticles. In the procedure, 34 mg of silicananoparticles after calcination were added to 8, 10 or 12 ml spinosad inethanal solution (1.7 mg/ml), with a spinosad:silica feeding ratio of0.4:1, 0.5:1 and 0.6:1, respectively (hereinafter denoted asnano-spinosad-X, where X is the ratio of spinosad:silica). The mixturewas removed into a long cylindrical flask attached to a rotaryevaporator (BUCHI R-210) and evaporated at 40° C. in a vacuum system indark with a residual pressure of 175 mbar until all solvent had beenremoved. For comparison purposes, a similar procedure was been carriedout with spinosad-ethanol solution only (no nano-particles beingpresent).

Characterization

The morphologies of the silica nanoparticles before and after theloading of spinosad were observed using and JEOL JSM 7800 field emissionscanning electron microscope (FE-SEM) operated at 0.8-1.5 kV. For FE-SEMmeasurements of pure silica nanoparticles, the samples were prepared bydispersing the powder samples in ethanol, after which they were droppedto the aluminium foil pieces and attached to conductive carbon film onSEM mounts. For FE-SEM measurements of nano-spinosad, the samples weredirectly attached to the conductive carbon film on SEM mounts.Transmission electron microscopy (TEM) images of the silica nanoparticeswere obtained with JEOL 2100 operated at 200 kV. For TEM measurements,the samples were prepared by dispersing and drying the powdersamples-ethanol dispersion on carbon film on a Cu grid. Fouriertransform infrared (FTIR) spectra were collected on a ThermoNicoletNexus 6700 FTIR spectrometer equipped with a Diamond ATR (attenuatedtotal reflection) Crystal. For each spectrum, 32 scans were collected atresolution of 4 cm-1 over the range 400-4000 cm-1. Wide angle X-raydiffraction (WA-XRD) patterns of the materials were recorded on a GermanBruker D8 X-ray diffractometer with Ni-filtered Cu Kα Radiation. AMetter Toledo GC200 thermogravimetric analysis (TGA) station was usedfor the loading amount and differential scanning calorimetry (DSC) studyat a heating rate of 2° C. min-1.

Release Test of Nano-Spinosad

In a release test, 2.67 mg nano-spinosad-X (containing 1 mg spinosad)was dispersed in 1 ml distilled water, respectively. The mixtures werekept at 25° C. incubator at 200 rpm. The supernatants were collected atdifferent time and the released amount of spinosad was measured andevaluated by using a UV-Vis spectrophotometer at a wavelength of 248 nm.The release amount of pure spinosad in water was also tested using thesame procedure.

UV-Stability Test of Nano-Spinosad

In this test, 1.2 mg pure spinosad and 4.2 mg nano-spinosad-0.4(containing 1.2 mg spinosad) were added in two transparent quartzcontainers, respectively. The using of quartz containers is to minimizedthe shielding of UV light from the containers. Each of the sample wasplaced under the UV light with a wavelength of 365 nm and power of 17.77mV cm⁻³. All samples were irradiated by the UV light for 2 hours. Afterthe irradiation, the spinosad and its degradation products wereextracted by acetonitrile (ACN) for three times and the finalconcentration was diluted to. 0.5 mg/ml dispersed in 1 ml distilledwater. The UV degradation conditions of both pure spinosad andnano-spinosad-0.4 were tested by high-performance liquid chromatography(HPLC) using ACN as the mobile phase.

In Vitro Bio-Assay

The in vitro effects of spinosad and nano-spinosad on the cattle tick,Rhipicephalus microplus were evaluated. The test was conducted atBiosecurity Sciences Laboratory (Queensland Government) using a standardLarval Immersion Test using organic solvent to extract the actives.

Preparation of MSHSs-RS

As shown in FIG. 1a MSHSs-RS particles were prepared by adding silicashell particles with smaller sizes (˜30 nm in diameter) onto MSHSs withrelatively larger sizes (˜400 nm). On the surface of MSHSs-RS particles,a void space between the small shell spheres with a radius of R1 (FIG.1a ) is generated for air entrapment. The air pocket is significantlyenlarged in these MSHS-RS particles because the internal sphericalcavity with a radius of R2 (R2>>R1) is connected with the air throughthe mesopores in the silica shell. The repulsion of the trapped air inthe void spaces towards water molecules provides the energy barrieragainst the wetting process because the hydroxyl groups in silica tendto absorb water molecules, as in the case of MSHSs (as shown in FIG. 1b). Therefore, the designed MSHSs-RS should demonstrate increasedhydrophobicity compared to MSHSs although both materials have the samepure silica composition. It is also advantageous compared to a solidnanoparticle with a rough surface because the solid nanoparticle with arough surface has less air pockets (no hollow core having a radius ofR2) and limited loading capacity of hydrophobic drugs. Previous studiesmainly focused on large flat surfaces; nanoparticles with hydrophiliccompositions and hydrophobic properties through surface roughnesscontrol have not been reported and have not been demonstrated forbio-applications. Images of the prepared MSHSs-RS were taken using ascanning electron microscope (SEM) and a transmission electronmicroscope (TEM) (see FIGS. 1c, 1e ). For comparison MSHSs with a smoothsurface were also prepared and characterised as shown in FIGS. 1d and 1f. Both nanoparticles have uniform and hollow spherical morphology withthe surface of MSHS-RS homogeneously decorated with silica shellparticles. In accordance with our theory, MSHSs-RS nanoparticles showunusual hydrophobic properties. Hydrophobicity was directly observed bythe dispersion of nanoparticles in a mixed solvent of water/diethylether. MSHSs-RS preferentially rests at the bottom of the diethyl etherlayer (a hydrophobic solvent) while MSHSs directly disperses in thewater layer. TGA profiles presented a small weight loss of 0.9% below200° C. for MSHSs-RS and 7.2% for MSHSs which can be attributed to theevaporation of moisture, indicating that the introduction of surfaceroughness makes MSHSs-RS more hydrophobic and thus it absorbs lessmoisture from the atmosphere than MSHSs.

To provide a quantitative comparison of the hydrophobicity betweenMSHSs-RS and MSHSs, a gel trapping technique (GTT) was employed andrevealed that MSHSs-RS have a contact angle value of 107.5°±10 whilstthat of MSHSs was 72.5°±5. The contact angle value of MSHSs-RS isslightly lower than that obtained for the octadecyltrimethoxysilanemodified silica (˜136°). Compared to MSHSs, MSHSs-RS exhibitsconsistently higher loading capacity for a range of hydrophobicmolecules, including RNase A (RNASE), insulin (INS), lysozyme (LYS), ahydrophobic dye, disperse red 1 (DR1) and a hydrophobic drug,griseofulvin (GRIS). These results further confirm that enhanced surfacehydrophobicity of MSHS-RS nanoparticles increases the loading capacityof hydrophobic molecules.

To test the adhesion of MSHSs-RS, animal fur was used as a model.MSHSs-RS and MSHSs with the same weight were dispersed in water andhomogeneously applied to two pieces of fur with the same size. Afterdrying and washing with water three times, the silica content remainingon fur was measured. FIG. 2A is the optical image of kangaroo fur thatillustrates typical hair structure. In comparison, pure silica showswhite particles under optical microscope due to its powder nature (FIG.2B). After application of silica nanoparticles onto the fur samples,white particles were observed attaching on the surface of the hairs,indicating the attachment of both silica nanoparticles (FIGS. 2C and2D). After three times washing, there are more white particles attachedonto the kangaroo hairs than in the case of MSHSs-RS (FIG. 2E) comparedto that of MSHSs-SS (FIG. 2F). This phenomenon indicates that MSHSs-RShave stronger adhesion ability on animal hairs. This conclusion is alsosupported from the ICPOES results. The silica weight percentageremaining on fur for MSHSs and MSHSs-RS was 28.5% and 51.0%,respectively. MSHSs-RS shows significantly improved adhesion due to itsrough surface and hydrophobicity. The enhanced adhesion of MSHSs-RSnanoparticles on fur should prolong the effective duration ofSpinosad-MSHSs-RS nano-formulation in field conditions.

A rotary evaporation method was utilized to encapsulate spinosad intosilica nanoparticles using spinosad-ethanol solution withspinosad:silica ratio of 0.4:1, 0.5:1 and 0.6:1. The nano-spinosadcomposites are denoted nano-spinosad-X where X stands for the ratio ofspinosad and silica. FIG. 3 shows the FTIR spectrum of pure spinosadwith obvious characteristic peaks at 891, 987, 1041, 1099, 1213, 1263,1371, 1456, 1660, 1707 and in the range of 2787-3012 cm⁻¹. The spectrumof silica nanoparticles shows a characteristic peak at 810 cm⁻¹ that canbe attributed to v(Si—O), and broad peak in the range of 1050-1200 cm⁻¹that can be attributed to —Si—O—Si bonding. In the spectra of allnano-spinsad-X, characteristic peaks 1371, 1456, 1660, 1707 and in therange of 2787-3012 cm⁻¹ can still be observed besides overlapping withthe characteristic peaks of silica. The FTIR spectra confirm thesuccessful encapsulation of spinosad with silica nanoparticles.

The actual loading amount of spinosad can be calculated by the weightloss from TGA results (FIG. 4A). Pure spinosad shows complete weightloss of 99.9% at 900° C. Pure silica nanoparticles after calcinationshows negligible weight loss from the adsorbed moisture (data no shown).The weight losses of nano-spinosad-X are 27.4, 32.8 and 38.1% for X=0.4,0.5 and 0.6, respectively. Accordingly, the loading amount ofnano-spinosad-X (X=0.4, 0.5 and 0.6) is calculated to be 28.6, 33.3 and37.5%, respectively, indicating that rotary evaporation can achievecomplete loading (˜100%) of spinosad.

The crystalline state of spinosad before and after encapsulation ischaracterized by WA-XRD (FIG. 5). The WA-XRD pattern of pure spinosadshows a series of sharp peaks in the range of 5-40°, indicating purespinosad is in a crystalline state. Pure silica nanoparticles show abroad peak centred at ˜22° which can be attributed to amorphous silica.Sharp characteristic peaks could not be observed in the WA-XRD patternof all samples of nano-spinosad-X beside the broad peak at 22° ofamorphous silica, indicating no crystalline spinosad is formed in thesesamples. The crystallization behaviour of nano-spinosad-X has also beenstudied by DSC (FIG. 4B). Pure spinosad displays a sharp endothermicpeak at 129° C. which indicates the melting point of crystallinespinosad. Similar to pure silica, all nano-spinosad-X show no obviouspeaks in the range of 25-350° C., indicating an amorphous state. Incomparison, a small endothermic peak at 129° C. is observed for thephysical mixture of spinosad and silica (pink), indicating the existenceof crystalline spinosad structure. The above results indicate thatspinosad was encapsulated into MSHS-RS nanoparticles in a nano-dispersedform by utilizing the rotary evaporation technique.

FE-SEM was used to directly observe the morphology of nano-spinosad(FIG. 6). The FE-SEM images show that silica nanoparticles areaggregated in low magnifications (FIG. 6A) and spherical morphology inhigh magnifications (FIG. 6B). If pure spinosad-ethanol solution is usedfor rotary evaporation, large crystalline spinosad with the size of ˜20μm (FIG. 6C) is formed. In the same magnification, all nano-spinosad-Xshow aggregations of small particles (FIG. 6D-FIG. 6F) which are exactlythe same morphology as pure silica without obvious crystals. Thesephenomena indicate the spinosad is successfully encapsulated in thecavity of MSHS-RS nanoparticles in different feeding ratios.

The release profile of both pure spinosad and nano-spinosad were testedin water. As shown in FIG. 7, for nano-spinosad, 16% of spinosad wasreleased in a short period time of 5 min and this level was maintaineduntil 540 min (release in water monitored by UV-Vis at 248 nm). On theother hand, for pure spinosad, only 2.4% of spinosad was released at 5min while the cumulative release is less than 8% even at 540 min.Spinosad confined in silica nanoparticles shows a solubility of ˜0.2mg/ml, which is more than two times higher than that of pure spinosad,similar to the solubility enhancement of curcumin confined insidemesoporous materials. Consequently, the release behaviour of spinosad isimproved compared to the pure spinosad. The fast release of a higherconcentration of spinosad is expected to be beneficial for thedevelopment of an “effective-immediately” nano-spinosad formulation.

The UV stability of nano-spinosad was studied. Both spinosad andnano-spinosad were irradiated under UV light (wavelength 365 nm, 17.77mV/cm³) for 2 h followed by HPLC which was used to monitor the productafter UV treatment utilizing ACN as the extraction media and mobilephase. As shown in FIG. 8, the peak at retention time of 3.5 min isattributed to spinosad. An additional peak at retention time of 1.5 minwas also observed in the pure spinosad group, which can be attributed tothe degraded product. This observation is in accordance with literaturereports, indicating spinosad itself is UV labile. However, in thenano-spinosad group, the degradation peak is not observed, suggestingthat the silica shell has a protective effect against UV irradiation forspinosad loaded inside the nano-cavity.

In order to confirm that the spinosad loaded into silica nanoparticlesis still effective, we evaluated the in vitro effects of spinosad andnano-spinosad on the cattle tick, Rhipicephalus microplus. The test wasconducted at Biosecurity Sciences Laboratory (Queensland Government)using a standard Larval Immersion Test. In the Larval Immersion Test,both spinosad and nano-Spinsad were firstly dissolved in organic solvent(2% Triton X-100 in acetone) to extract the actives for stock solution(10 mg/ml) and then be diluted in water. Both spinosad and nano-spinosadshow dose-dependent mortality to cattle tick larval. Three assays wereconducted to narrow the LC ranges. In Assay 3, the spinosad shows LC50and LC99 value to larval cattle ticks of 54 and 196 ppm in 24 h,respectively. Nano-spinosad shows LC50 and LC99 value to larval cattleticks of 46 and 159 ppm in 24 h, respectively. These results indicate ofnano-spinosad show comparable and slightly better toxicity to ticklarval models. This result confirms that after encapsulation thespinosad loaded in silica nanoparticles is still effective.

This example shows that MSHSs-RS show prolonged adhesion behaviour toanimal fur. Using a rotary evaporation method, spinosad can be loadedinto such hollow MSHS-RS nanoparticles with ˜100% loading. The loadingamount can reach up to 28.6-37.5% (wt/wt) as determined by TGA analysis,while WA-XRD and DSC analysis confirmed that spinosad was dispersed inthe nano-cavity of the MSHS-RS in an amorphous state. Consequently, therelease behaviour of spinosad is improved compared to the pure spinosad.Furthermore, the silica shell has a protective effect against UVirradiation for spinosad loaded inside the nano-cavity thus providingUV-shielding and protection of the labile spinosad. The nano-spinsadafter loading in the cavity of the MSHS-RS is proved to be comparablyeffective to cattle tick larval (Rhipicephalus microplus). With enhancedwater solubility, UV stability and fur adhesion of Spinosad-MSHSs-RS,this nanoformulation is expected to have prolonged duration of efficacyunder field conditions.

Example 2—Forming MSHS-RS

This example describes an embodiment of the method of the third aspectof the invention for forming MSHS-RS nanoparticles.

The procedures for controlled synthesis of monodispersed rough silicahollow spheres are schematically illustrated in FIG. 9. In a typicalStöber synthesis condition, resorcinol (0.15 g) and formaldehyde (0.21mL) were added into a basic solution with 70 mL of ethanol, 10 mL ofwater and 3 mL of ammonia (28 wt %) with a pH about 11.5 to formresorcinol-formaldehyde (RF) nanospheres at room temperature with adiameter of 180 nm after 6 h of polymerization. These RF nanosphereswill form a sacrificial particle that will be eventually removed. Then,a certain amount of tetraethyl orthosilicate (TEOS) was added into thereaction solution, followed by another addition of resorcinol andformaldehyde 5 minutes later in Step II. Due to the difference betweensilica and RF deposition rates in Step II, a triple-layered shell wasformed on the preformed RF core spheres. Specifically, a relativelydense silica layer was firstly deposited on the surface of preformed RFspheres, because of the faster condensation speed of silica oligomerscompared with RF oligomers. Following passage of a certain time, whenthe RF oligomers started to polymerize, the intergrowth of RF along withthe silica species started on the surface of the first silica layer,followed by a preferentially vertical growth of these two species. Thisresulted in the formation of hybrid second layer of ‘rod-like’ silicaand RF. With the consumption of silica species, the remaining RFoligomers further deposited on the second layer to form an outmost layerof pure RF. By adjusting the amount of TEOS from 1.4 to 0.6 mL added inStep II, the thickness of the first silica layer reduced and thedistance between the ‘rod-like’ silica enlarged due to the decreasingcondensation rate of silica species. It should be noted that, with only0.6 mL of TEOS added, the first silica layer is not continuous with somecrevices existed. This may result from the discrete distribution ofsilica nuclei on the pre-formed RF surface and slow growth beforemerging together to form a relatively continuous interlinked silicalayer. After calcination in air in Step III, the RF species in thehybrid composites were removed, leaving the silica hollow spheres withrough surface. The final silica products are denoted as S-1.4, S-1.0 andS-0.6 where the number represents the volume amount of TEOS addition inStep II.

The representative transmission electron microscopy (TEM) images ofS-1.4, S-1.0 and S-0.6 are shown in FIGS. 10A-10C. Monodispersed silicahollow sphere with rough surface were observed in all the samples. Theaverage particle size of S-1.4, S-1.0 and S-0.6 is estimated to be 300,280 and 250 nm, respectively. The hollow cavity size of three samples isalmost the same at about 160 nm, which is relatively smaller than thesize of preformed RF nanospheres (180 nm). This may be caused byshrinkage during calcination. The ‘rod-like’ rough structure on theshell can be clearly identified from the TEM images, and a decreasingdensity of silica ‘rod’ distribution on the shell can also be revealed.Dynamic light scattering (DLS) analysis was further utilized todetermine the particle size and monodispersity. As shown in FIG. 10D,the hydrodynamic diameter of S-1.4, S-1.0 and S-0.6 is about 295, 310and 325 nm, respectively, which is slightly larger than those determinedby TEM due to surrounded water molecules. The narrow particle sizedistribution curves with a small polydispersity index (PDI) value(0.053, 0.086, and 0.101 for S-1.4, S-1.0 and S-0.6 respectively)indicate all of the silica hollow spheres possess uniform particle sizesand excellent monodispersity.

As shown in the TEM images shown in FIG. 10, the ‘rod-like’ roughstructure can be clearly identified, however, the first silica layerbeneath it is hardly revealed, even though a higher contrast appearedinside of the silica shell. To further explore the detailed structuresof those rough silica hollow spheres, an electron tomography (ET)technique was utilized by taking a tilted series from +70° to 70° withan increase step of 1°. The tomograms were obtained by processing thetilted images with 10 nm Au fiducial alignment via IMOD. The tomogramslices referring to the middle part of the rough silica hollow spheresare shown as FIGS. 11 A-C. The silica shell observed from TEM imagesactually was composed of two layers, one relatively dense silica layerand another rough layer with ‘rod-like’ structures. The thickness of thedense silica layer decreased from 41 nm in S-1.4 to 31 nm in S-1.0, andeven 19 nm in S-0.6. The decreasing thickness may result from the slowersilica condensation rate with less TEOS amount addition. Interestingly,the relatively dense silica shell in S-1.4 (FIG. 11 A) and S-1.0 (FIG.11 B) are both continuous without any pore structures connecting thehollow cavity. However, the one in S-0.6 showed several crevices with awidth about 1-2 nm distributed on this layer (FIG. 11 C, black arrows),which provided transport channels for small molecules to access theinner hollow space.

To further characterize this ‘rod-like’ structure and its distributionon the hollow sphere surface, a quantitative comparison was conducted.Due to the similar particle size of these rough hollow spheres, thedistance between each silica ‘rod’ can indirectly represent itsdistribution density. Even though the interstitial geometry between thesilica ‘rods’ is different from the traditional pore structures, itsspace can also be revealed by nitrogen sorption analysis and its poresize distribution (Ref Langmuir 1999, 15:8714; J. Colloid InterfaceScience, 2008, 317:442). As shown in FIG. 12A, the nitrogen adsorptionand desorption results of these rough silica hollow spheres exhibited atype IV isotherm, with a hysteresis loop between 0.5-1.0 of P/P₀,indicating the existence of mesopore structures on the hollow spheresurface. The pore size distribution by BJH adsorption branch as shown inFIG. 12B indicated the distance between each silica ‘rod’ was enlargedfrom about 6.3 nm in S-1.4 to 7.5 nm in S-1.0, and to 9.3 nm in S-0.6.With enlarged distance between the silica ‘rods’, there are more of thespaces provided for the nitrogen molecules to condense, which willfinally achieve a higher amount for adsorption (Ref Langmuir 1999,15:8714). This is in accordance with the surface area and pore volumeincrease from 133 m²/g and 0.19 cm³/g of S-1.4, to 167 m²/g and 0.26cm³/g of S-1.0, and 182 m²/g and 0.37 cm3/g of S-0.6 with enlargedinterstitial distance.

The introduction of surface roughness on various substrates has beenachieved by a biomimetic approach in both microscale and nanoscale. Thesurface roughness results in enhanced hydrophobicity notwithstandingthat the silica from which the nanoparticles are made is normallyhydrophilic. However, the traditional characterization approach of waterdroplet contact angle results cannot be easily related to the contactangle of the individual particles, especially in nanoscale. Hence, a geltrapping technique (GTT), which is based on the proportional entrappingof individual nanoparticles on the oil-water surface, has been developed[Ref: Langmuir, 2004, 20:9594]. By spreading the particles on anoil-water surface and subsequent gelling of the water phase, thenanoparticles trapped on the water phase are then replicated and liftedup with poly(dimethylsiloxane) (PDMS) elastomer, which allows theparticles partially embedded in the PDMS surface to be imaged with SEM[Ref Langmuir 2003, 19:7970]. This method allows the quantitativecomparison of the surface hydrophobicity for individual nanoparticleswith different surface roughness to correlate the interstitial distanceof the rough structures and its hydrophobicity. To justify the enhancedsurface hydrophobicity by rough structures, a smooth silica hollowsphere (FIG. 5A insert), with relatively dense silica shell thickness of60 nm and no obvious mesopores on the shell, was selected as a control.As shown in FIG. 5, the smooth particle exhibited a contact angle ofonly 61°, while with rough structure emerged on the surface, the contactangle increased to 73° for S-1.4 and 86° for S-1.0. With even largerinterstitial distance, the contact angle can reach 102°, indicating ahydrophilic silica material equipped with hydrophobic surface by theintroduction of roughness, and increasing of surface hydrophobicity ofthe particles with enlarged distance between silica ‘rods’.

To show the usefulness of the rough surface hollow silica spheres forthe take-up of hydrophobic material, the rough silica hollow sphereswere used for lysozyme adsorption. For comparison, smooth silica hollowsphere was employed as a control group, which only achieved 82 μg/mg.For the rough silica hollow spheres, an obvious enhancement for lysozymeadsorption was observed in FIG. 6, with the capacity for S-1.4 and S-1.0increased to 358 and 408 μg/mg, respectively. Especially for S-0.6, theadsorption capacity can even reach as high as 641 μg/mg. The increasingadsorption capacity should be attributed to the larger interstitialdistance, rising pore volume and enhanced surface hydrophobicityintroduced by the surface roughness, as well as the volume provided bythe accessible hollow central cores of the spheres.

Example 3—Delivery Systems for Use in Biological Systems

In biological systems, hydrophobic interactions are usually consideredto be the strongest of all long-range non-covalent interactions.Hydrophobic interaction is beneficial for adsorption of biomolecules,improving interaction with cellular membranes increasing the uptake ofnanoparticles for cellular delivery as well as tailoring the releaserate of drugs. To generate nanoparticles with hydrophobic properties,the choices of hydrophobic composition or functionalization are amongthe convenient approaches. Hydrophobic material such as carbon nanotubes(CNTs) have shown great promise as nanoscaled vehicles for drugdelivery, however one of the main concerns is the fact that CNTs couldbe hazardous to environment and human health which need further surfacefunctionalization to reduce their intrinsic toxicity. Hydrophobicmoieties such as alkanethiols and alkyl chains have been modified ontothe surfaces of various nanoparticles including gold and silica toenhance the loading of hydrophobic drugs/protein and improve cellulardelivery performance. However, chemically grafted hydrophobic groupstended to cause unwanted toxicity and pore blocking of nano-carriers. Itis therefore a challenge to design a safe and efficient nanocarriersystem employing an alternative approach.

In this example, surface roughness engineering was achieved by addingsilica shell particles with smaller sizes (˜13 or 30 nm in diameter)onto mesoporous hollow spheres of silica (MHS) with relatively largersizes (˜200 or 400 nm). The surface roughening creates the voids (thespace between small shell spheres with a radius of R1) on the outersurface for air entrapment. The air pocket is significantly enlarged inthis design because the internal spherical cavity with a radius of R2(R2>>R1) is connected with the air through the mesopores in the silicashell. The repulsion of the trapped air in the void spaces towards watermolecules provides the energy barrier against the wetting processbecause the hydroxyl groups in silica tend to adsorb water molecules asin the case of mesoporous hollow silica (MHS). Compared to rough solidStöber (RSS) silica nanoparticles, rough mesoporous hollow spheres ofsilica (RMHS) provide more space to trap the air, leading to a higherenergy barrier during the wetting process and thus more distinguishedhydrophobicity. The nature of hydrophilic composition of RMHS provides ahigh loading capacity of the ‘last resort’ antibiotic vancomycin (VAN)while the hydrophobic property facilitates the controlled release of VANand adhesion to bacteria, resulting in enhanced antibacterial efficacy,compared to free VAN and MHS-VAN.

MHS nanoparticles were synthesized using a surfactant-directing alkalineetching strategy. RMHS was prepared by mixing positively charged MHSafter amino group functionalization with negatively charged Stöbersilica nanoparticles (˜40 nm in diameter) followed by calcination.Scanning electron microscope (SEM) and high resolution transmissionelectron microscopy (HRTEM) images show that RMHS of 450 nm in averagesize have been successfully prepared with a uniform spherical morphology(FIG. 14a, 14c ). The surfaces of RMHS are homogeneously decorated with40 nm silica nanospheres, indicating the successful attachment of silicananospheres to the surface of MHS. In contrast, MHS (FIG. 2b, 2d ) hasan average particle size of 350 nm. HRTEM images (FIG. 14c, 14d )clearly indicate the hollow core@porous shell structure of RMHS and MHS.The hollow core is ˜230 nm in diameter and the porous shell is about 60nm in thickness. SEM images show the hollow core of the nanoparticleswith monodisperse morphology for both MHS and RMHS. The hydrodynamicsize of MHS and RMHS was further measured by dynamic light scattering(DLS), which shows a size of 396 nm for MHS and 459 nm for RMHS,consistent with both SEM and TEM results. The distance between twoneighboring silica nanospheres is measured at around 30 nm and the gapbetween them provides space for the air entrapment.

Both MHS and RMHS exhibited typical type-IV isotherms with an H2-typehysteresis loop, indicating the existence of well-defined mesopores. Thepore size distributions calculated from the adsorption branches usingthe Barrett-Joyner-Halenda (BJH) method showed that both samples haveuniform mesopores centered at 3.5 nm. RMHS has a relatively lowersurface area compared to MHS (342 vs. 427 m² g⁻¹) because the shellparticles are solid. The higher pore volume of RMHS (0.46 vs 0.31 cm³g⁻¹ of MHS) is mainly attributed to the inter-particle packing voids asreflected by the capillary condensation step which occurred at relativepressure (P/P₀) higher than 0.95. Surface charge measurement byz-potential showed that both RMHS and MHS were negatively charged to asimilar degree. Both samples have pure silica in composition asconfirmed by Fourier transform infrared spectroscopy (FTIR), showingcharacteristic peaks for physisorbed water (—OH) at 1620 cm⁻¹, silanolgroup (Si—OH) at 790 cm⁻¹, as well as siloxane group (Si—O—Si) at 1062and 449 cm⁻¹.

The hydrophobicity of the nanoparticles was observed by the dispersionof MHS and RMHS in a mixed solvent of water/diethyl ether. RMHSpreferentially rests at the bottom of the diethyl ether layer (ahydrophobic solvent) while MHS directly disperses in the water layer (ahydrophilic solvent) even without gentle shaking. RSS was also showingsimilar behavior as RMHS in the water/diethyl ether layer. This occurreddue to the competition between the affinity of Si—OH towards watermolecules and the repulsion of the trapped air in void spaces towardsboth oil and water molecules (as presented in FIG. 1).Thermalgravimetric analysis (TGA) profiles presented a small weight loss of0.9% below 200° C. for RMHS while 7.2% for MHS which can be attributedto the evaporation of moisture. The TGA results indicate that theintroduction of surface roughness makes RMHS more hydrophobic and thusit absorbs less moisture from the atmosphere than MHS.

A dye (rose Bengal, RB) adsorption method was also employed toquantitatively determine the relative hydrophobicity of nanoparticles. Aplot of partitioning quotient (PQ) versus nanoparticle surface area permillilitre was constructed for RMHS, MHS and RSS. The slope of this plotis proportional to the relative hydrophobicity of nanoparticles.Compared to RSS with the slope of 0.000675×10⁻⁹ mL μm⁻², RMHS yieldedhigher slope value of 0.00106×10⁻⁹ mL μm², indicating higherhydrophobicity of RMHS compared to RSS. MHS on the other hand showed thelowest slope with no significant value suggesting a hydrophilic nature.

RMHS and MHS were used in the adsorption of three hydrophobic proteinsincluding RNase A (RNASE), insulin (INS) and lysozyme (LYS) ahydrophobic dye, disperse red 1 (DR1) and a hydrophobic drug,griseofulvin (GRIS). As shown in FIG. 15a , a higher loading capacitywas achieved exclusively for five sorbates by RMHS than MHS. Compared toMHS, a faster adsorption rate of DR1 (FIG. 15b ) and LYS was alsoobserved when comparing RMHS to MHS. These results indicate thatenhanced surface hydrophobicity of nanoparticles favours higher andfaster loading of hydrophobic molecules. The loading capacity of RSStowards LYS was also measured as 25.9 mg g⁻¹. Compared to that of RMHS(263.1 mg/g), the much lower LYS loading amount of RSS can be attributedto its solid nature.

To further understand the role of air which induced RMHS hydrophobicity,the adsorption capacity of RMHS towards LYS was conducted in solutionsafter removing air bubbles under a vacuum condition. The adsorptionamount of the LYS on RMHS was found to be reduced by 37.3% (from 263.1mg g⁻¹ to 172.1 mg g⁻¹), comparable with the adsorption capacity of MHS(161.5 mg g⁻¹). An additional experiment was conducted by eliminatingthe pre-sonication process to retain most of the air trapped by thenanoparticles. Higher loading of LYS was achieved by RMHS withoutsonication with 27.5% increment compared to the adsorption using RHMSsubject to pre-sonication steps in FIG. 15a . These results confirmedthe role of air as the hydrophobic solvent on the RMHS structure whichsubsequently improves the adsorption for protein. In contrast, surfaceroughness has no influence on the loading capacity of a hydrophilicmolecule, VAN, as shown in FIG. 4c . Similar loading value was achievedfor both MHS and RMHS with this hydrophobic molecule. However, thehydrophobic property of RMHS enabled sustained release behaviour of VANup to more than 36 h relative to 100% release at 8 h for MHS (FIG. 15c).

The size of the core nanoparticles with similar morphology can befurther finely tuned with the same preparation method. The inventorshave successfully prepared MHS and RMHS with an average core size of 200nm and 13 nm shell particles size named as MHS200 and RMHS200. Bothnanoparticles have similar surface morphology compared to the largerparticles (MHS and RMHS) as shown by TEM and SEM images. MHS200 andRMHS200 have slightly smaller pore size (3.4 nm) and relatively higherpore volume (0.38 cm³ g⁻¹ for MHS200 and 0.62 cm³ g⁻¹ for RMHS200)compared to the larger sized particles.

The use of nanoparticles as a delivery vehicle for antibiotics providesa promising strategy through prolonged drug circulation half-life,increased availability of drugs interacting with membrane molecules andpromoted sustained drug release. VAN is an antibiotic useful for thetreatment of a number of bacterial infections since it inhibits the cellwall synthesis in susceptible bacteria. To demonstrate the antibacterialefficacy of VAN delivered by the surface engineered materials, drugloaded nanoparticles were incubated with Escherichia coli (E. coli).Nanoparticles with a size of 200 nm (MHS200 and RMHS200) were chosen inthis study because the screen test showed that compared to largerparticles (˜400 nm), smaller ones exhibited higher bacterial toxicityeffect. The in vitro antibacterial activity of VAN, MHS200-VAN andRMHS200-VAN was evaluated by monitoring the optical density (OD) at 600nm of a bacterial suspension. E. coli (1×10⁶ CFU mL⁻¹) was incubated inLuria-Bertani (LB) medium in a 1.5 ml centrifuge tube at variousconcentrations of VAN for 18 h. The minimum inhibitory concentration(MIC) value of free VAN towards E. coli was observed at 25 μg mL⁻¹ (FIG.16a ). This value reduced to 20 μg ml⁻¹ for RMHS200-VAN which is lowerthan the dosage used with VAN conjugated MCM-41 (200 μg ml⁻¹) inin-vitro E. coli culture at 18 h. In a separate experiment, MHS200-VAN,RMHS200-VAN and free VAN with the same VAN content of 25 μg ml⁻¹ wereincubated with 1×10⁶ CFU mL⁻¹ E. coli in LB media and OD was measured asa function of time. It was observed that RMHS200-VAN maintained 100%inhibition throughout 24 h. However, re-growth of bacteria as evidencedby increases in OD was observed in both MHS200-VAN and free VAN groupsafter 18 h (FIG. 16b ).

It was reported that the re-growth of bacteria exposed to VAN may occurif inadequately inhibited bacteria synthesize new peptidyglocan tooverride the antibacterial effect of VAN. The 100% inhibition of E. colieven at 24 h in the case of RMHS200-VAN should be attributed to twoadvantages coming from the nanoparticle design: 1) the rough surfaceparticles have a higher efficacy compared to their smooth counterparts;and (2) the hydrophobic nature of RMHS200 which leads to a sustainedrelease of VAN compared to MHS200 (FIG. 16d ), similar to the largersized nanoparticles (FIG. 16c ). Eventually the effective time window ofthe drug is increased.

To provide direct evidence on the antibacterial efficacy ofnanoparticles, TEM was employed to observe the morphology of E. colicultured at 24 h (FIG. 16(c-f)). For the untreated group (FIG. 16c ),the typical cylindrical morphologies of E. coli remained intact.Compared to the untreated group, VAN treated bacteria showed damage ofthe bacterial membrane (FIG. 16d-f ). For MHS200-VAN, MHS200 was foundin the bacterial membrane (FIG. 16e ) and severe damage of thewall/membrane of E. coli (FIG. 16f ) was clearly observed. The cellcytotoxicity of MHS200 and RMHS200 to normal human dermal fibroblast(HDF) was also assessed by the MTT assay. No significant cytotoxicity ofboth nanoparticles even at a concentration of up to 500 μg/mL wasobserved, providing evidence of excellent bio-inertness and safety ofthe materials as the carrier system.

In conclusion, this example shows that the inventors have successfullyprepared novel nanoparticles with a hydrophilic silica composition buthaving hydrophobic properties through surface roughness modification,which show higher loading capacity of hydrophobic molecules andsustained release for hydrophilic drugs compared to their counterpartswith a smooth surface. The fundamental understanding gained from thisstudy provides a new strategy for the development of nanocarriers withsafe composition and high performance in widespread drug deliveryapplications.

Example 4—Preparation of Carbonaceous Nanoparticles

In this example, a new sequential heterogeneous nucleation (SHN) pathwayto prepare self-organized colloidal carbon nanoparticles withcontrollable mesostructures and morphologies in the absence of structuredirecting agents is reported. The SHN concept is schematicallyillustrated in FIG. 17. The synthesis is carried out in an ethanol/watersystem with NH₃.H₂O as the catalyst, simply using tetraethylorthosilicate (TEOS), resorcinol and formaldehyde (RF) as precursors. Instep I when TEOS and RF precursors are mixed together, Stöber spheresare formed through a homogenous nucleation process due to the relativelyfaster condensation rate compared to the RF system. Once the silicaspheres are formed, the RF precursors preferentially condense on thesilica surface through heterogeneous nucleation. In order to tune thewall structure, TEOS is introduced again in step II, which formsuniformly distributed silica nanoparticles on the RF shell surfacethrough a subsequent heterogeneous nucleation process. The residual RFoligomers further condense on the top of silica nanoparticles to createa second RF layer. After carbonization with or without hydrothermaltreatment (step III) under inert atmosphere followed by the removal ofsilica (step IV), mesostructured hollow carbon spheres (MHCS) with abilayered structure are obtained. By controlling the thickness ofcarbon/silica shells, the bilayered morphology (invaginated,endo-invaginated or intact spheres) and mesopore size can be finelyregulated.

From the scanning electron microscopy (SEM) images presented in FIG. 18(A,C), MHCS prepared without hydrothermal treatment in step III exhibitan invaginated spheroidal morphology, much like a deflated balloon whereone side of the sphere becomes enfolded towards the other. Transmissionelectron microscopy (TEM) images of the invaginated MHCS show a clearlybilayered and hollow internal structure (FIG. 18B). When MHCS areprepared with hydrothermal treatment, an intact spheroidal morphology isobtained as shown by the SEM image (FIG. 18C). TEM observations forthese particles also show a bilayered concentric spherical structure(FIG. 18D).

Invaginated and intact MHCS exhibit uniform outer diameters of 250 and270 nm, respectively. Moreover, both particles disperse well in aqueoussolution and produce the characteristic Tyndall effect commonly observedfor monodispersed colloidal suspensions (FIG. 18E). Dynamic lightscattering (DLS) measurements reveal a hydrated particle size of 265 and295 nm for invaginated and intact MHCSs, respectively (FIG. 18F). Thenarrow size distributions and low polydispersity index (PDI of 0.1) fortwo samples indicate both MHCS possess highly uniform particle size andexcellent water dispersibility. High resolution SEM images reveal highlyporous, rough external surfaces with open-pore entrances for theinvaginated MHCS. Intact MHCS on the other hand, exhibit relativelysmooth and continuous surface morphology.

For three-dimensional (3D) nano-objects with complex internal structuressuch as MHCS, investigation by conventional TEM may provide misleadinginformation. This is because TEM images provide the collectivestructural information over a certain thickness and merge it into a 2Dprojection. For example, the fine structures between the inner and outerlayers are not clear. Moreover, it seems that two spheres shown in FIG.18B (indicated by arrows) are not invaginated, although this effectcould result from the electron beam passing perpendicular to the planeof invagination. Electron tomography (ET) is a rapidly developingtechnique for the advanced 3D imaging of complex structures, whichallows virtual reconstruction of a material's internal structure using3D models built from a series of 2D slices (19, 20).

We used the ET technique to study the detailed structures of MHCS. Aseries of tilted images was taken in the range of +70 to −70° withincrements of 1°. Using this technique, one can clearly observe theinvaginated MHCS particle apparently changing from an invaginated to anintact spherical structure. This highlights the ambiguity of the dataprovided by conventional TEM alone and confirms the importance of ETcharacterization for materials with complex and asymmetricalarchitectures. To observe the detailed internal structures of MHCS,electron tomograms were generated from two perpendicular tilting seriesusing IMOD software (21). The ET slice which cuts perpendicular to theinvagination face of the MHCS (FIG. 19A) exhibits a clearly bilayered,crescent moon-like morphology. The inner and outer layers are linked bythin carbon bridges of approximately 1-2 nm in thickness (indicated byblack arrows). In contrast, a tilt-series of the intact MHCS reveals acomplete spherical morphology throughout the rotation (data not shown).The ET slide in FIG. 19C shows a full moon-like morphology for theintact MHCS, where the two concentric layers are linked by moresubstantial carbon bridges with approximately 4-5 nm in thickness.

The invaginated and intact samples also differ noticeably in thicknessand the degree of continuity of inner and outer shells. The outer layersof the invaginated and intact samples appear relatively continuous withan average thickness of 6 and 12 nm respectively, however the innerlayer of the invaginated structure shows numerous defects andinterruptions which form a more fragile and discontinuous inner shellwhen compared to the intact structure. The average sizes of the voidspaces between two layers measure approximately 15 and 20 nm radiallyfrom the inner to the outer shell for the invaginated and intact MHCS,respectively. Digitally reconstructed structures for two MHCS with innershells in orange and outer shells in yellow are shown in FIGS. 19B and19D, respectively. Invagination of both the inner and outer shells canbe observed for the invaginated MHCS while spherical morphology is seenfor the intact MHCS, which is consistent with the morphologicalobservations from TEM and SEM. Moreover, carbon bridges linking theinner and outer shell can also be observed for both invaginated andintact MHCS.

Nitrogen sorption studies for both invaginated and intact MHCS showtype-IV adsorption isotherms. The BJH pore sizes calculated from theadsorption branch indicate pore sizes of 15.9 and 18.0 nm for theinvaginated and intact MHCS, respectively. These pore sizes correspondclosely with the measured interlayer distance between the inner andouter shells observed in ET and TEM micrographs, suggesting thisconfined interlayer space is responsible for the BJH pore sizedistribution. The BET surface area and pore volume of invaginated MHCS(1032 m2 g-1 and 2.11 cm3 g-1, respectively) are slightly higher thanthose obtained for the intact MHCS (880 m2 g-1 and 1.44 cm3 g-1,respectively), which may be attributed to thinner shells and thus theincrease in bulk-to-surface ratio for the more solidly constructedintact MHCS. The X-ray photoelectron spectra (XPS) show that only peaksfrom C1s (˜285 eV) and O1s (˜534 eV) are detected, revealing the majorcomponents of both invaginated and intact MHCSs are carbon and oxygen(22). The mass percentage of carbon and oxygen are calculated to be92.9% and 7.1%, respectively. The X-ray diffraction (XRD) patternsreveal the amorphous nature of MHCS.

In order to understand the formation mechanism of MHCS, wesystematically studied the nucleation and growth processes of silica-RFparticles as a function of time. Since both TEOS and RF canindependently polymerize under the same conditions to form uniform solidparticles (FIG. 20, curve I and II), their individual reaction kineticswas first investigated. Under the synthesis conditions utilised, thepolymerization of TEOS results in formation of silica particles within15 minutes (m), consistent with the typical induction period commonlyobserved in Stöber sphere formation (23). These spheres then rapidlyincrease in size up to 2 h, after which particle size is relativelyconsistent. RF polymerization under the same conditions on the otherhand, forms spheres with slower growth. The formation of some irregularRF polymer nucleates is observable at 1 h, which continue developinginto spherical particles by 2 h. The RF spheres increase in sizerelatively rapidly from 2 to 6 h followed by a slower growth region till12 h. From curve II it can be inferred free RF oligomers persist in thesynthetic system at 12 h.

When TEOS and RF are added simultaneously, FIG. 20 curve III revealsthat the particle size initially (up to 1 h) follows the same trend asthe pure silica system with only silica particles are formed. After 2 hthe particle sizes increase gradually to 250 nm at 12 h, formingsilica@RF core-shell structures with increasing RF shell thickness as afunction of time. No evidence of solid RF spheres nor solid carbonspheres after carbonization/silica etching can be found, indicating thatthe RF polymerization system has been changed from homogeneous toheterogeneous nucleation on the surface of silica cores, consistent withclassical nucleation theory that the free energy barrier forheterogeneous nucleation on a surface is considerably lower as comparedto homogeneous nucleation. However, this approach leads to hollow carbonspheres with only microporous walls, which has little control over themorphology and mesostructures of final products and thus limitedapplications.

When TEOS is introduced in step II at a carefully chosen time-point of 6h, TEM was used to monitor the structural evolution over the following 2h. From TEM images of samples after calcination in air, it can be seenthat a secondary population of silica nucleus appears on the surface ofsilica@RF particles within 15 m after the second TEOS addition. Thesecondary silica nanoparticles increase in size from ˜5 nm at 15 m up to˜10 nm at 30 m before merging together to form a relatively continuousinterlinked silica shell with a radial thickness of 18 nm at 2 h. Aftersecondary TEOS addition, the particle size steadily increases (FIG. 20,curve IV) relative to the silica@RF particles shown in curve III,achieving an additional 30 nm in diameter after 12 h of growth. TEM dataconfirm the absence of any solid silica nanoparticles in the finalproducts. The above observations indicate that the RF layer of silica@RFparticles formed in step I triggers a subsequent heterogeneousnucleation of TEOS. Due to the slower polymerization behavior of RFsystem, the remaining RF precursors preferentially nucleate on silicasurface. The sequential heterogeneous nucleation of two polymerisablesystems and their interplay gives rise to an interpenetrating silica-RFcomposite shell structure. Removing silica in the core and shell aftercarbonization results in the final structures of MHCS.

The ET results of fine structures of MHCS are in accordance with the SHNmechanism. The bridges in between two carbon layers come from theintergrowth of RF with secondary silica nanoparticles. Hydrothermaltreatment favors further condensation of RF system, leading to thickerbilayers as well as bridges and eventually intact MHCS. The invaginatedMHCS with exposed porous surface are formed due to the thinner RF layersand bridges when hydrothermal treatment is not used in step III.

The SHN mechanism can recur over additional nucleation cycles. As ademonstration, a third addition of silica and RF precursors wasintroduced to the system. The resulting triple-layered MHCS structuresare consistent with another cycle of heterogeneous nucleation. The addedTEOS heterogeneously nucleates on the RF surface, forming an additionalpopulation of silica nanoparticles, followed by heterogeneous nucleationand growth of RF over silica. The use of SHN pathway under the samepolymerization conditions for multiple cycles provides scope for thedesign of nanomaterials with elegant structures.

Judicious selection of time-points for the addition of TEOS in step IIcan determine the form of the final structures. When TEOS was addedearlier (at 3 h time point instead of 6 h), no obvious bilayeredstructures were observed for both the invaginated and intact carbonparticles. Instead, the structures exhibit single layered mesoporouscarbon shells. When TEOS was added at 24 h, only hollow microporouscarbon structures with thickness of 15 nm are obtained. These resultsdemonstrate that carefully controlling the polymerization kinetics andelaborately regulating the nucleation process of TEOS and RF precursorsin sequence enables the formation of bilayered MHCS.

To investigate the parameters influencing the invagination of hollowparticles, we prepared a series of single layered hollow carbon sphereswith controlled wall thicknesses. Wall thickness was controlled from 5to 16 nm via the increase in stirring time from 6 to 36 h (step I in thescheme). The results clearly demonstrate that when the thickness ofsingle layered hollow carbon sphere is as thin as 5 nm, most particlesshow invaginated morphology. With an increased thickness to 8 nm, only asmall number of invaginated spheres can be observed, while an increaseto 13 nm yields only intact spheres. This study demonstrates that thethickness of carbon layer plays a crucial role in controlling theinvaginated or intact morphologies of the final products.

The distance between the shells was tuned from 7 to 27 nm by increasingthe amount of TEOS from 0.5 to 2.5 ml added in step II. All the samplesprepared without hydrothermal treatment exhibit invaginated morphologywhile the samples with hydrothermal treatment exhibit intact sphericalmorphology. The pore sizes, BET surface areas and pore volumes of thesamples calculated from N₂ sorption are consistent with the resultsobtained from TEM measurements. The general trend is that the sampleswithout hydrothermal treatment exhibited much higher surface areas andpore volume than those with hydrothermal treatment, consistent with whatwe observed before. Moreover, the greater the distance between theshells, the higher the observed surface area and pore volume. This canbe ascribed to the enlarged mesoporous interlayer region in samples withlarge interlayer spacing. The corresponding silica templates showincreased sizes and continuity of silica shells with the increasingamount of TEOS added.

Notably, when the interlayer spacing is enlarged to 27 nm and afterhydrothermal treatment, an unprecedented structure with the inner layerinvaginated while the outer layer remains intact (so calledendo-invaginated structure) is obtained (FIG. 21A). FIGS. 20B and 20Cshow two TEM images recorded along x- and z-axis (parallel andperpendicular to endo-invaginated plane), respectively. The ET sliceshown in FIG. 21D reveals the cross-sectional crescent and sphericalmorphology of the inner and outer layers respectively along the yz planeright in the middle of the endo-invaginated structure. Two additional ETslices are given along the xy plane (FIGS. 21E and 21F) at z-height of aand b as indicated in FIG. 21A, respectively, showing two concentricrings and three concentric rings accordingly. Some carbon bridges can beobserved connecting the outer-most ring to the middle ring (FIGS. 21Eand 21F). The middle ring however, has no observable bridges connectingthe inner-most ring, indicating that these two surfaces originallycoming from the inner sphere are not fused. These distinct structurefeatures would be impossible to obtain using conventionalcharacterization techniques other than ET.

The invagination of the inner shell can be ascribed to the formation ofa thick and continuous silica layer during step II, which limits theinterpenetration of RF and thus decreases the thickness and density ofthe carbon bridges. It is also noted that shell thickness of the outersphere is thicker compared to that of the inner invaginated one (FIGS.21D-21F) attributed to the hydrothermal treatment. With reduced supportfrom bridging between the outer and inner shell, the more fragile innersphere with thinner walls partially detaches and collapses away from thethicker, intact outer shell, forming the unique endo-invaginated MHCS.

We further tested the application of MHCS for lysozyme adsorption. Forboth invaginated and intact particles, around 75% of the saturationadsorption can be achieved within 10 minutes, suggesting fast adsorptionkinetics towards lysozyme. The maximum adsorbed amount of lysozyme onthe invaginated particles is around 1250 μg mg⁻¹ after 6 h, showing thehighest adsorption capacity towards lysozyme compared to previousreports. The fast adsorption rate and high adsorption capacity should beattributed to the large entrance size, high surface area and thehydrophobicity of the invaginated MHCS.

This example demonstrates that colloidal carbon particles withunprecedented structures (invaginated, endo-invaginated and intactbilayered morphologies) have been designed via a sequentialheterogeneous nucleation pathway through the self-organization of twopolymerizable systems. This SNH mechanism defines the recurringheterogeneous nucleation cycles through which nanostructuredinterpenetrating composites can be self-organized and the structure,morphology of colloidal carbon nanoparticles can be precisely adjusted.

Example 4: Demonstration of Enhanced Adhesion to Bacterial Cell Walls

E. coli, a typical gram-negative bacteria, was employed and incubatedwith silica hollow spheres (concentration of 100 μg·mL⁻¹) in Luria Broth(LB) media. The particle-bacteria adhesion of MSHS-SS and MSHS-RSparticles was compared to demonstrate the effect of the rough silicasurface by direct observation using electron microscopy after bacteriafixation and staining. As shown in FIG. 22 a, E. coli exhibits intactrod-like morphology with fewer MSHS-SS particles adhered to thebacterial surface compared to MSHS-RS-B (FIG. 22b ) and MSHS-RSparticles (FIG. 22c ). To be noted, some of MSHS-RS particles arepartially engulfed into the bacteria cell wall, leaving a semi-sphericaldent on the bacteria surface upon detachment (FIG. 22c , identified byblack arrows). The engulfment process is typically related to thestrength of the attractive cell membrane-particle interactions, anindication of enhanced adhesion between MSHS-RS particles and thebacteria cell wall. In contrast, the smooth surface of MSHS-SS particlesprovides limited contact area for interfacial interaction, resulting inless particles adhered on bacteria surface. Moreover, the electrostaticrepulsion between both negatively charged silica nanoparticles andbacteria surface hinders their interaction as well. It is favorable toenhance the electrostatic attraction towards bacteria for silicananoparticles by amine modification. However, the unwanted toxicityinduced by the amine groups remains a concern. Here, by engineeringsurface roughness, MSHS-RS particles show enhanced bacterial adhesionproperties, which may be attributed to the multivalent interactionsinduced by their surface spikes when contacting with the hairy bacteriasurface, resulting in strong adhesion via a large number of contacts.

To quantitatively analyze the silica amount adhered on the bacteriasurface, the bacteria cultured with the silica particles were filteredthrough a 450 nm-pore filter membrane. Extensive washing was applied toremove the isolated particles in the solution. Bacteria-free sampleswere also analyzed as a control and to eliminate the interference fromaggregated silica particles. The ICP results (FIG. 22d ) show that lessthan 0.1 pg of MSHS-SS particles adhere on each bacterial cell surface,whereas, 0.36 pg of MSHS-RS-B and 0.48 pg of MSHS-RS particles remain oneach bacteria.

Example 5: Formulation with Lysozyme

To demonstrate delivery efficiency of the silica particles withantimicrobial agents, lysozyme was immobilized in these silica hollowspheres. As shown in FIG. 23a , due to the limited external surface areaprovided for lysozyme adsorption, MSHS-SS particles show the lowestloading capacity of only 61 μg·mg⁻¹ (μg lysozyme per mg of silica). Incontrast, MSHS-RS particles exhibit the highest loading capacity of 270μg·mg⁻¹, which is two times of that achieved by MSHS-RS-B particles (135μg·mg⁻¹). This is attributed to the increase of mesopore volume from0.117 cm³·g⁻¹ (MSHS-RS-B) to 0.229 cm³·g⁻¹ (MSHS-RS). The surface zetapotential of silica hollow spheres before and after lysozyme loading wascharacterized in 10 mM phosphate buffer solution (PBS). After lysozymeloading, zeta potential of MSHS-SS particles changes dramatically from−29 mV to −3 mV, indicating the positive charged lysozyme is adsorbed onthe external surface. However, for MSHS-RS-B and MSHS-RS particles,their surface charge change from −19 mV and −18 mV to −8 mV and −6 mV,respectively. This suggests that lysozyme molecules are typicallyimmobilized into the mesopores of the MSHS-RS-B and MSHS-RS particles,resulting in limited neutralization of surface charge.

Example 6: Lysozyme Release

Lysozyme release behaviour from the silica particles was examined underthe condition with fixed initial lysozyme concentration (270 μg·mL-1) inPBS. MSHS-SS particles exhibit a boost release of lysozyme with morethan 85% released within 18 h. Compared to these smooth particles,MSHS-RS-B particles show a relatively slower release rate with around75% of lysozyme released after 24 h. MSHS-RS particles exhibit the mostsustained release profile among three particles, with only 74% oflysozyme released at 72 h. However, MSHS-RS with a relatively large poresize are supposed to have a fast release profile. The retarded releaseof protein molecules from MSHS-RS may result from the enhanced surfacehydrophobicity induced by the surface roughness and accessible innercavity.

Example 7: Antibacterial Activity of Formulated Lysozyme

The in vitro antibacterial activity of free lysozyme and lysozyme loadedsilica particles formulated using the above procedure were compared bythe optical density (OD) measurement. E. coli (5×10⁶ CFU·mL⁻¹) wasincubated with various concentrations of lysozyme and correspondinglysozyme loaded silica particles for 24 h. Across all samples dosedependent antibacterial performance was observed wherein higherconcentrations/loadings of lysozyme resulted in greater antibacterialactivity. Lysozyme formulated into the silica particles showed higheractivity compared to free lysozyme at the same lysozyme concentrationand this effect is more significant at lysozyme concentration above 500μg·mL⁻¹. Rough silica particles exhibit enhanced antibacterial activitytowards E. coli relative to free lysozyme and MSHS-SS particlesespecially for MSHS-RS particles, showing a minimum inhibitoryconcentration (MIC) value of 700 μg·mL⁻¹ for the latter. In contrast,the MIC of free lysozyme towards E. coli cannot be achieved even at theconcentration as high as 2 mg·mL⁻¹.

To further demonstrate the advantages of the silica particles aslysozyme carriers, the long-term bacterial inhibition was tested viabacteria kinetic tests under batch culture. The time dependent bacterialgrowth at lysozyme concentration of 700 μg·mL⁻¹ was monitored for 3 days(FIG. 2b ). LB-agar plate assay was employed to examine the bacterialviability after 3-day treatment. It was observed that MSHS-RS particlesmaintained 100% bacterial inhibition throughout the three day test. Thisthree-day inhibition result is comparable to the performance of silverloaded silica nanoparticles at 80 μg·mL⁻¹ as demonstrated by thebacterial kinetic assay. In contrast, time dependent bacterial growth asevidenced by the increase of OD value is observed for MSHS-SS, MSHS-RS-Band free lysozyme formulations. No viable colonies can be observed onthe agar plates for bacteria treated with lysozyme loaded MSHS-RSparticles showing strong bactericidal activity of the silica particlesas opposed to the other samples. The long-term bacterial inhibitionproperty should be attributed to two advantages provided by the designof the silica particles: 1) enhanced adhesion to bacterial surfaceenabled by the surface roughness which results in efficient, targeteddelivery of lysozyme and enriched local concentration of lysozyme on thebacterial surface, and 2) prolonged antimicrobial activity achieved bythe sustained release of lysozyme from MSHS-RS particles. However, dueto relatively weak particle-bacteria interaction and fast lysozymerelease, MSHS-SS and MSHS-RS-B fail to control the bacterial growth withinadequate lysozyme concentration delivered efficiently towards thebacterial surface.

Example 8: Formulation of Particles with Ivermectin

Ivermectin was loaded using rotary evaporation into the MSHS-RS, MSHS-SSand MSHS-RS particles functionalised with hydrophobic octadecyl moietiesto render the surface more hydrophobic. Thermogravimetric analysis (TGA)showed an ivermectin loading level in the silica particles of around 23wt. %, which is in accordance with the feeding ratio of ivermectin tosilica nanoparticles (1:3).

To investigate the UV protection properties of the silica particlestoward ivermectin these nano-formulations as well as pure (free)ivermectin were treated under UV irradiation for 3 h. The samples beforeand after UV irradiation were analysed using high performance liquidchromatography (HPLC) to identify their compositions. Free ivermectinwas fully degraded after 3 h of irradiation. The ivermectin loaded intoMSHS-SS particles showed significant degradation of ivermectin. This mayresult from the fact that ivermectin is only partially loaded into theinternal cavity of the MSHS-SS particles, resulting in only partialprotection. In contrast, HPLC analysis ivermectin formulations usingMSHS-RS particles and hydrophobically modified MSHS-RS particles showedno significant degradation of ivermectin, indicating the ivermectincomposition was well protected by the nanoparticles.

Example 9: Varying the Size of MSHS-RS Particles

MSHS-RS nanoparticles with different particle size were synthesized byvarying the amount of resorcinol and formaldehyde in the first addition.By increasing the resorcinol and formaldehyde amount, larger RF polymernanospheres can be formed acting as the core, leading to an increase inMSHS-RS particle diameters from 307 nm (resorcinol 0.15 g) to 564 nm(resorcinol 0.3 g), to 704 nm (resorcinol 0.45 g) and to 837 nm(resorcinol 0.6 g). As shown in FIG. 24, the resulting MSHS-RS particlesin varied sizes still maintain the spiky surface topography. Nitrogensorption results showed that the resulting particles has mesoporousstructures with pore size around 10-20 nm. As the particle sizeenlarges, the surface area and pore volume of the MSHS-RS particlesincreased as shown in Table 1.

TABLE 1 Nitrogen sorption determined physicochemical properties forMSHS-RS particles. Samples S_(BET) (m²/g) V_(Total) (cm³/g) d_(pore)(nm) resorcinol 0.15 g 178 0.434 12.1 resorcinol 0.30 g 227 0.548 16.5resorcinol 0.45 g 268 0.665 16.5 resorcinol 0.60 g 275 0.831 16.5

Example 10: Formulation with p-DNA

MSHS-RS and MSHS-SS particles with a diameter of around 350 nm weresynthesized. Highly negative charges are a well-recognised feature ofp-DNA molecules, thus cationic functional groups were introduced ontothe silica particles to enhance the electrostatic attraction betweenp-DNA and the silica by coating the silica particles withpolyethylenimine (PEI). After PEI modification, the silica particlesstill maintain their spiky topography. Nitrogen sorption results showedthat PEI modified MSHS-RS particles exhibited mesoporous structures withpore size around 11 nm. The pore size can be enlarged to 16 and 19 nm byhydrothermal treatment at temperatures of 100 and 130° C. respectively,and the hydrothermal treatment at 150° C. can further enlarge the poresize with a wide distribution from 20 to 80 nm. The zeta potential ofthese the MSHS-RS particles changes from negative (˜−20 to −30 mV) topositive (˜+15 mV) after PEI modification, indicating the successfulintroduction of PEI groups on the silica particle surface.

Nanodrop measurement and a gel retardation assays were performed toassess binding capacity with the plasmid pcDNA3-EGFP that encodes forEnhanced. Green Fluorescent Protein (EGFP). PEI modified MSHS-RSparticles display much higher pcDNA3-EGFP binding capacity (29.7 ng/μg)than the PEI-modified MSHS-SS particles (14.7 ng/μg). To be noted,MSHS-RS particles that has undergone hydrothermal treatment showed evenlarger p-DNA loading capacity compared with the MSHS-RS particleswithout hydrothermal treatment due the enlarged pore size and porevolume. In the gel retardation assay, a constant amount of pcDNA3-EGFP(0.5 μg) was mixed with various amounts of PEI modified silica particlesfrom 0 to 80 μg.

Example 11: Formulation of Battery Electrodes and Battery Cells

SeS₂ was impregnated into carbon particles in accordance with thepresent invention by a simple melt-impregnation to obtain theSeS₂/carbon composite. A transmission electron microscope (TEM) image ofSeS₂/carbon is shown in FIG. 25b . It is clearly seen that the contrastis higher in the interlayer space than in the hollow cavity. Thisdifference is not observed in the TEM image of the bare particles (FIG.25a ), indicating that SeS₂ predominately locates in the interlayerspace between the two carbon shells rather than in the cavity. Theunderlying reason is possibly due to a higher capillary force in asmaller nano-space to attract SeS₂, which explains our observation intrial experiments that single-layered carbon hollow spheres withmicroporous walls cannot load S/SeS₂ in their cavity. Therefore, thechoice of multi-layered hollow carbon such as the carbon of the presentinvention is essential in our design. The electrochemical evaluationsuggests that the SeS₂/carbon composite exhibits an excellent cyclingstability, high specific capacity and high Coulombic efficiency (FIG.25c ). A battery was constructed using the SeS₂/carbon particles as thebasis of the cathode and lithium metal was used as the anode. After 100cycles, the reversible capacity still remains at 930 mAh/g with nocapacity decay at 200 mA/g. The Coulombic efficiency levels off at 99.5%from the 2^(nd) cycle. For comparison, pure SeS₂ shows a much inferiorcycling performance. The capacity decreases continuously throughout thecycling such that after 100 cycles a low capacity of 75 mAh/g isobserved. These proof-of-concept results highlight that carbon spheresof the present invention are excellent hosts for SeS₂ and theSeS₂/carbon composites are promising electrode materials fornext-generation Li—SeS_(x) batteries.

In the present specification and claims (if any), the word ‘comprising’and its derivatives including ‘comprises’ and ‘comprise’ include each ofthe stated integers but does not exclude the inclusion of one or morefurther integers.

Reference throughout this specification to ‘one embodiment’ or ‘anembodiment’ means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearance of the phrases ‘in one embodiment’ or ‘in an embodiment’ invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more combinations.

In compliance with the statute, the invention has been described inlanguage more or less specific to structural or methodical features. Itis to be understood that the invention is not limited to specificfeatures shown or described since the means herein described comprisespreferred forms of putting the invention into effect. The invention is,therefore, claimed in any of its forms or modifications within theproper scope of the appended claims (if any) appropriately interpretedby those skilled in the art.

1. Particulate material comprising rough mesoporous hollownanoparticles.
 2. Particulate material as claimed in claim 1 wherein therough mesoporous hollow nanoparticles comprise a mesoporous shell, theexternal surface of which has projections thereon, the projectionshaving smaller sizes than the particle size.
 3. Particulate material asclaimed in claim 2 having a particle size ranging from 100 nm to 3000nm, a size of the projections ranging from 5 nm to 1000 nm. 4.Particulate material as claimed in claim 2 wherein the size of theprojections ranges from 100 nm to 500 nm.
 5. Particulate material asclaimed in claim 2 wherein the projections comprise nanospheres on theshell or outgrowths on the shell.
 6. Particulate material as claimed inclaim 2 wherein the mesoporous shell comprises silica, Ag, Au, calciumphosphate or titanium dioxide or carbon or a carbon-based material. 7.Particulate material as claimed in claim 1 wherein the rough mesoporoushollow nanoparticles comprise rough mesoporous hollow silicananoparticles.
 8. Particulate material as claimed in claim 1 wherein theparticles are made from a material that is normally hydrophilic but theparticles demonstrate hydrophobic characteristics.
 9. Particulatematerial as claimed in claim 2 wherein the rough mesoporous hollownanoparticles have a hollow core having a diameter of from 100 nm to1000 nm.
 10. Particulate material as claimed in claim 2 wherein thehollow core is defined by a shell having a mesoporous structure. 11.Particulate material as claimed in claim 10 wherein the shell has a porestructure that includes pores in the range of from 2 nm to 20 nm. 12.Particulate material as claimed in claim 2 wherein the shell thatsurrounds the hollow core has a thickness of from 10 nm to 100 nm. 13.Particulate material as claimed in claim 2 comprising projections oroutgrowths on the surface of the shell, the projections or outgrowthsbeing spaced apart from each other and providing surface roughness tothe particles.
 14. Particulate material as claimed in claim 1 comprisingrough mesoporous hollow silica nanoparticles having a hollow core havinga diameter of from 100 nm to 1000 nm, the hollow core being defined by asilica shell having a mesoporous structure, the silica shell having apore structure that includes pores in the range of from 2 nm to 20 nm,the silica shell that surrounds the hollow core having a thickness offrom 10 nm to 100 nm, the rough mesoporous hollow silica nanoparticleshaving silica projections or outgrowths on the surface, spaced apartfrom each other to provide surface roughness to the particles. 15.Particulate material as claimed in claim 2 wherein the spacedprojections comprise nanoparticles connected to the outer surface of alarger hollow nanoparticle.
 16. Particulate material as claimed in claim15 wherein the nanoparticles connected to the outer surface of thelarger hollow nanoparticles are of a same composition as the largerhollow nanoparticles or of a different composition to the larger hollownanoparticles.
 17. Particulate material as claimed in claim 15 whereinthe nanoparticles used to construct the projections have a diameter inthe range of from 5 nm to 100 nm and the hollow nanoparticles have adiameter in the range of from 100 nm to 3000 nm.
 18. Particulatematerial as claimed in claim 2 wherein the projections comprise spacedprojections comprising strands or cylinders or fibres or nodulesextending outwardly from the hollow shell of the nanoparticles. 19.Particulate material as claimed in claim 18 wherein the length of theprojections is from 5 nm up to the diameter of the large hollow particleon which they reside.
 20. Particulate material as claimed in claim 18wherein a diameter of the projections ranges from 2 nm up to 100 nm.21-94. (canceled)